Ionizable compounds and compositions and uses thereof

ABSTRACT

This invention includes ionizable compounds, and compositions and methods of use thereof. The ionizable compounds can be used for making nanoparticle compositions for use in biopharmaceuticals and therapeutics. More particularly, this invention relates to compounds, compositions and methods for providing nanoparticles to encapsulate active agents, such as nucleic acid agents, and to deliver and distribute the active agents to cells, tissues, organs, and subjects.

BACKGROUND OF THE INVENTION

Therapeutic agents such as drug compounds, nucleic acid molecules andother active agents operate by uptake into cells, tissues, and organs ofa subject. Transfection of agents and molecules into cells is often alimiting step in therapeutic action.

When the active agent molecules are sensitive to attack or degradationin serum or other biological settings, it becomes necessary to protectthe molecules in order to achieve their medicinal effect.

For example, one way to carry out transfection of nucleic acids is toencapsulate the active molecules in a lipid nanoparticle. Drawbacks ofthis methodology include potential toxicity in various modalities ofdelivery, such as intravenous injection, and low rates of cellpenetration.

There is a long-standing need for molecules to provide nanoparticlesthat have favorable transfection properties to deliver active agents tocells.

What is needed are compositions and compounds for forming nanoparticlesfor active agents. There is a continuing need for lipid-like moleculesand compositions for efficient transfection and distribution of nucleicacid molecules and other agents to cells and subjects.

BRIEF SUMMARY

This invention relates to molecules and compositions thereof for use inbiopharmaceuticals and therapeutics. More particularly, this inventionrelates to compounds, compositions and methods for providingnanoparticles to deliver and distribute active agents or drug compoundsto cells, tissues, organs, and subjects.

This invention provides a range of ionizable compounds. The ionizablecompounds of this invention can be used to form nanoparticles to deliverand distribute active agents.

Embodiments of this invention include a broad range of compounds havinglipid-like or liposome-forming properties.

In some embodiments, a compound may have the structure shown in FormulaI

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, and aminoalkyl;each R⁷ is independently selected from H, alkyl, and hydroxyalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;Q is O or NR⁷;p is from 1 to 4.

In further embodiments, a compound can have the structure shown inFormula II

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group; wherein R³ is a C(12-20) alkyl group or a C(12-20)alkenyl group that is substituted with a carboxylic acid or ester group.

In additional embodiments, a compound may have the structure shown inFormula III

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is selected from alkyl, hydroxyalkyl, alkoxyalkoxy, andcarboxyalkyl;wherein R⁶ is selected from NR⁷ ₂, N⁺HR⁷ ₂ and N⁺R⁷ ₃;wherein R⁷ is selected from H, alkyl, hydroxyalkyl.

A compound of this invention may have the structure shown in Formula IV

wherein R¹ and R² areR¹=C(═O)OR⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;Q is O or NR⁷;p is from 1 to 4.

In further embodiments, a compound can have the structure shown inFormula IV-B

wherein R¹ and R² areR¹=C(═O)OR⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein Z is S or O;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;p is from 1 to 4.

In further aspects, a compound may have the structure shown in Formula V

wherein R¹ and R² areR¹=NHC(═O)R⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein p is from 1 to 4;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;Q is O or NR⁷.

A compound of this invention can have the structure shown in Formula VI

wherein R¹ isR¹=OC(═O)R⁴wherein R² and R⁴ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from aminoalkyl, quaternary aminoalkyl.

In certain embodiments, a compound of this invention can have thestructure shown in Formula VII

wherein R¹ and R² areR¹=OC(═O)R⁴R²=OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is selected from H, alkyl, aminoalkyl, quaternary aminoalkyl,hydroxyalkyl, alkoxyalkyl, alkoxyalkoxyalkyl.

In further aspects, a compound may have the structure shown in FormulaVIII

wherein R¹ and R² areR¹=OC(═O)R⁴R²=C(═O)ZR⁵wherein Z is NH or O,wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

amino;

quaternary amino;

aminoalkyl;

quaternary aminoalkyl;

NHC(═O)(CH₂)_(p)R¹⁰;

NHC(═O)SR⁹;

wherein R¹⁰ is selected from

carboxyalkyl;

aminoalkyl;

wherein R⁹ is selected from

alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

and wherein

each R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;

each R⁷ is independently selected from H, alkyl, hydroxyalkyl;

each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;

q is from zero to four;

Q is or NR⁷.

In certain aspects, a compound may have the structure shown in FormulaVIII-B

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2;R⁴ and R⁵ are independently for each occurrence a C(12-20) alkyl group,or a C(12-20) alkenyl group;wherein Z is N, O;wherein R³ is selected from alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy,aminoalkyl;

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;p is from 1 to 4.

In additional embodiments, a compound can have the structure shown inFormula IX

wherein R¹ and R² areR¹=C(═O)OR⁴R²=NHC(═O)R⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein p is from 1 to 4;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;Q is O or NR⁷.

A compound of this disclosure can have the structure shown in Formula X

wherein R¹ and R² areR¹=C(═O)OR⁴R²=NHC(═O)R⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

amino;

quaternary amino;

aminoalkyl;

quaternary aminoalkyl;

hydroxyalkylamino.

In further embodiments, a compound may have the structure shown inFormula XI

wherein R¹ and R² areR¹=C(═O)R⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein Z is O or NH;wherein p is from 1 to 4;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;Q is O or NR⁷.

This invention further contemplates compositions containing an ionizablecompound above and a pharmaceutically acceptable carrier. In someembodiments, the composition may contain nanoparticles. This disclosureincludes pharmaceutical compositions comprising an ionizable compoundabove, an active agent, and a pharmaceutically acceptable carrier. Theionizable compound may be from 15 mol % to 40 mol % of the lipids of thecomposition. In some embodiments, the composition may comprisenanoparticles.

An active agent of this disclosure may be one or more RNAi molecules. Anactive agent may be one or more RNAi molecules selected from smallinterfering RNAs (siRNA), double stranded RNAs (dsRNA) that are Dicersubstrates, microRNAs (miRNA), short hairpin RNAs (shRNA), DNA-directedRNAs (ddRNA), Piwi-interacting RNAs (piRNA), repeat associated siRNAs(rasiRNA), and modified forms thereof.

An active agent of this disclosure may be one or more activepharmaceutical ingredients.

In certain embodiments, this invention includes compositions for use indistributing an active agent for treating a condition or disease in asubject, the composition comprising an ionizable compound above, astructural lipid, a stabilizer lipid, and a lipid for reducingimmunogenicity of the composition. The active agent can be one or moreRNAi molecules and the composition may comprise nanoparticles thatencapsulate the RNAi molecules.

This invention further contemplates compositions containing an ionizablecompound, and one or more pharmaceutically acceptable excipients. Insome embodiments, a composition of this invention can be a nanoparticlecomposed, at least in part, of an ionizable compound.

Compounds of this invention can be used to make compositions for use indistributing an active agent in a subject, where the compositionincludes an ionizable compound.

A composition of this invention can be used in distributing an activeagent for treating a condition or disease in a subject.

A composition for use in distributing an active agent for treating acondition or disease in a subject can include an ionizable compound, astructural lipid, and a lipid for reducing immunogenicity of thecomposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scheme for the preparation of Compound A6.

FIG. 2 shows a scheme for the preparation of Compound AB.

FIG. 3 shows a scheme for the preparation of Compound A4.

FIG. 4 shows a scheme for the preparation of Compound B8.

FIG. 5 shows a scheme for the preparation of Compound A9.

FIG. 6 shows a scheme for the preparation of Compound AA.

FIG. 7 shows a scheme for the preparation of Compound A5.

FIG. 8 shows a scheme for the preparation of Compound A1.

FIG. 9 shows a scheme for the preparation of Compound D22.

FIG. 10 shows a scheme for the preparation of Compounds A7 and A8.

FIG. 11 shows a scheme for the preparation of Compounds C3 and C2.

FIG. 12 shows a scheme for the preparation of Compound DD.

FIG. 13 shows a scheme for the preparation of Compound E4.

FIG. 14 shows a scheme for the preparation of Compound CA.

FIG. 15 shows a scheme for the preparation of Compound D1.

FIG. 16 shows a scheme for the preparation of Compound D7.

FIG. 17 shows a scheme for the preparation of Compound F6.

FIG. 18 shows a scheme for the preparation of Compounds F5 and F7.

FIG. 19 shows a scheme for the preparation of Compounds F8 and F9.

FIG. 20 shows a scheme for the preparation of Compounds C25 and C24.

FIG. 21 shows a scheme for the preparation of Compound D16.

FIG. 22 shows a scheme for the preparation of Compound D17.

FIG. 23 shows a scheme for the preparation of Compound D18.

FIG. 24 shows a scheme for the preparation of Compound D19.

FIG. 25 shows a scheme for the preparation of Compound D20.

FIG. 26 shows a scheme for the preparation of Compound D21.

FIG. 27 shows a scheme for the preparation of Compound E37.

FIG. 28 shows a scheme for the preparation of Compounds E38 and E39.

FIG. 29 shows a scheme for the preparation of Compound E40.

FIG. 30 shows a scheme for the preparation of Compound A23.

FIG. 31 shows a scheme for the preparation of Compound A24.

FIG. 32 shows a scheme for the preparation of Compound A25.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a range of ionizable molecules that areamphiphiles with lipid-like properties. The ionizable compounds of thisinvention can be used in delivering therapeutic agents to cells, tissuesor organs, organisms, and subjects.

In some aspects, this invention provides compounds for forming lipidnanoparticles for encapsulating and delivering active agents such asnucleic acid molecules to cells and subjects.

This invention can provide a composition for use in distributing anactive agent in cells, tissues or organs, organisms, and subjects, wherethe composition includes one or more of the ionizable molecules of thisinvention.

Compositions of this invention may include one or more of the ionizablemolecules, along with a structural lipid, and one or more lipids forreducing immunogenicity of the composition.

An ionizable molecule of this invention can be any mol % of acomposition of this invention.

Compositions of this invention may include one or more of the ionizablemolecules, along with a structural lipid, and one or more lipids forreducing immunogenicity of the composition.

Compositions of this invention may include one or more of the ionizablemolecules, along with a structural lipid, one or more stabilizer lipids,and one or more lipids for reducing immunogenicity of the composition.

This invention includes compositions containing one or more ionizablecompounds and a pharmaceutically acceptable carrier. The composition maycomprise nanoparticles.

This invention further contemplates pharmaceutical compositionscontaining one or more ionizable compounds, an active agent, and apharmaceutically acceptable carrier.

A composition may contain an ionizable compound in a quantity from 15mol % to 40 mol % of the lipids of the composition.

An active agent can be one or more RNAi molecules. An active agent canbe one or more RNAi molecules selected from small interfering RNAs(siRNA), double stranded RNAs (dsRNA) that are Dicer substrates,microRNAs (miRNA), short hairpin RNAs (shRNA), DNA-directed RNAs(ddRNA), Piwi-interacting RNAs (piRNA), repeat associated siRNAs(rasiRNA), and modified forms thereof.

In further aspects, this invention includes compositions for use indistributing an active agent for treating a condition or disease in asubject, where the composition contains one or more ionizable compounds,a structural lipid, a stabilizer lipid, and a lipid for reducingimmunogenicity of the composition. The active agent can be one or moreRNAi molecules, and the composition may comprise nanoparticles thatencapsulate the RNAi molecules.

Compositions with Three Components

As used herein, a component of a formulation, such as a “lipid,” can bea single compound, or can be a combination of one or more suitable lipidcompounds. For example, “a stabilizer lipid” can refer to a singlestabilizer lipid, or to a combination of one or more suitable stabilizerlipids. One skilled in the art can readily appreciate that certaincombinations of the compounds described herein can be used without undueexperimentation, and that various combinations of compounds areencompassed by the description of a component of a formulation.

The ionizable compounds of a composition of this invention can be from50 mol % to 80 mol % of the lipid components of the composition. Incertain embodiments, the ionizable molecules of a composition can befrom 55 mol % to 65 mol % of the lipid components of the composition. Infurther embodiments, the ionizable molecules of a composition can beabout 60 mol % of the lipid components of the composition.

The structural lipid of a composition of this invention can be from 20mol % to 50 mol % of the lipid components of the composition. In certainembodiments, the structural lipid of a composition can be from 35 mol %to 45 mol % of the lipid components of the composition.

The one or more lipids for reducing immunogenicity of the compositioncan be from a total of 1 mol % to 8 mol % of the lipid components of thecomposition. In certain embodiments, the one or more lipids for reducingimmunogenicity of the composition can be from a total of 1 mol % to 5mol % of the lipid components of the composition.

In additional aspects, a composition of this invention can furtherinclude a cationic lipid, which can be from 5 mol % to 25 mol % of thelipid components of the composition. In certain embodiments, acomposition of this invention can further include a cationic lipid,which can be from 5 mol % to 15 mol % of the lipid components of thecomposition. In these aspects, the molar ratio of the concentrations ofthe cationic lipid to the ionizable molecules of a composition of thisinvention can be from 5:80 to 25:50.

In compositions of this invention, the entirety of the lipid componentsmay include one or more of the ionizable compound molecular components,a structural lipid, and one or more lipids for reducing immunogenicityof the composition.

Compositions with Four Components

The ionizable molecules of a composition of this invention can be from15 mol % to 40 mol % of the lipid components of the composition. Incertain embodiments, the ionizable molecules of a composition can befrom 20 mol % to 35 mol % of the lipid components of the composition. Infurther embodiments, the ionizable molecules of a composition can befrom 25 mol % to 30 mol % of the lipid components of the composition.

The structural lipid of a composition of this invention can be from 25mol % to 40 mol % of the lipid components of the composition. In certainembodiments, the structural lipid of a composition can be from 30 mol %to 35 mol % of the lipid components of the composition.

The sum of the stabilizer lipids of a composition of this invention canbe from 25 mol % to 40% mol % of the lipid components of thecomposition. In certain embodiments, the sum of the stabilizer lipids ofa composition can be from 30 mol % to 40 mol % of the lipid componentsof the composition.

In some embodiments, a composition of this invention can include two ormore stabilizer lipids, where each of the stabilizer lipids individuallycan be from 5 mol % to 35 mol % of the lipid components of thecomposition. In certain embodiments, a composition of this invention caninclude two or more stabilizer lipids, where each of the stabilizerlipids individually can be from 10 mol % to 30 mol % of the lipidcomponents of the composition.

In certain embodiments, the sum of the one or more stabilizer lipids canbe from 25 mol % to 40 mol % of the lipids of the composition, whereineach of the stabilizer lipids individually can be from 5 mol % to 35%mol %.

In certain embodiments, the sum of the one or more stabilizer lipids canbe from 30 mol % to 40 mol % of the lipids of the composition, whereineach of the stabilizer lipids individually can be from 10 mol % to 30%mol %.

The one or more lipids for reducing immunogenicity of the compositioncan be from a total of 1 mol % to 8 mol % of the lipid components of thecomposition. In certain embodiments, the one or more lipids for reducingimmunogenicity of the composition can be from a total of 1 mol % to 5mol % of the lipid components of the composition.

In additional aspects, a composition of this invention can furtherinclude a cationic lipid, which can be from 5 mol % to 25 mol % of thelipid components of the composition. In certain embodiments, acomposition of this invention can further include a cationic lipid,which can be from 5 mol % to 15 mol % of the lipid components of thecomposition. In these aspects, the molar ratio of the concentrations ofthe cationic lipid to the ionizable molecules of a composition of thisinvention can be from 5:35 to 25:15.

In certain embodiments, the entirety of the lipid components of acomposition may include one or more of the ionizable compound molecularcomponents, a structural lipid, one or more lipids for reducingimmunogenicity of the composition, and one or more stabilizer lipids.

Examples of Lipid Compositions

In some embodiments, three lipid-like components, i.e. one or moreionizable molecules, a structural lipid, and one or more lipids forreducing immunogenicity of the composition can be 100% of the lipidcomponents of the composition. In certain embodiments, a cationic lipidcan be included.

Examples of compositions of this invention are shown in Table 1.

TABLE 1 Compositions of lipid components (each in mol % of total)Ionizable Cationic Structural Reduce immun. 60 0 32 8 60 0 35 5 55 0 441 65 0 32 3 60 0 36 4 65 0 32 3 70 0 25 5 74 0 20 6 78 0 20 2 50 10 35 555 15 25 5 55 20 20 5

In certain embodiments, four lipid-like components, i.e. one or moreionizable molecules, a structural lipid, and one or more lipids forreducing immunogenicity of the composition, and one or more stabilizerlipids can be 100% of the lipid components of the composition.

Examples of compositions of this invention are shown in Table 2.

TABLE 2 Compositions of lipid components (each in mol % of total)Ionizable Cationic Structural Stabilizer Reduce immun. 17 0 35 40 8 20 035 40 5 25 0 35 39 1 25 0 35 35 5 25 0 30 40 5 25 0 40 30 5 30 0 25 40 535 0 25 35 5 40 0 30 25 5 25 5 30 35 5 25 10 30 30 5 25 15 25 30 5

Compositions for Selective Biodistribution

Aspects of this invention can provide a range of compositions for use indistributing an active agent to various organs or tissues of a subject.

For example, compositions of this invention can contain an ionizablelipid, a structural lipid, and a lipid for reducing immunogenicity ofthe composition.

In some embodiments, compositions of this invention can contain anionizable lipid, a structural lipid, one or more stabilizer lipids, anda lipid for reducing immunogenicity of the composition.

Compositions of this invention may provide a surprisingly selectivebiodistribution of the active agent to a particular organ or tissue.

In some embodiments, a composition of this invention can provide asurprisingly selective biodistribution of the active agent to the lungof a subject.

In further embodiments, a composition of this invention can provide asurprisingly selective biodistribution of the active agent to the liverof a subject.

In some embodiments, a composition of this invention can provide asurprisingly selective biodistribution of the active agent to the colonof a subject.

In some embodiments, a composition of this invention can provide asurprisingly selective biodistribution of the active agent to thepancreas of a subject.

In certain embodiments, the ratio of the distribution of the activeagent to the lung over the distribution of the active agent to thesubject's liver can be at least 1.5.

In further embodiments, the ratio of the distribution of the activeagent to the lung to the distribution of the active agent to thesubject's liver can be at least 5.

Ionizable Compounds

The ionizable compounds of this invention can have lipid-likeproperties, for example, as amphiphiles.

Examples of an ionizable molecule include compounds having the structureshown in Formula I

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is selected from 1-azetidines, 1-pyrrolidines, 1-piperidines,4-morpholines, and 1,4-piperazines wherein the rings can be substitutedat any carbon atom position,

and can also be selected from amino and aminoalkyl groups, which may besubstituted,

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, and aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;Q is O or NR⁷;p is from 1 to 4.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(16-18) alkyl group, or a C(16-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, p is 1, 2, 3 or 4.

In some embodiments, q is 0, 1, 2, 3 or 4.

In some embodiments, examples of an ionizable molecule include compoundshaving the structure shown in Formula I

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;Q is or NR⁷.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, examples of an ionizable molecule include compoundshaving the structure shown in Formula I

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl;Q is O or NR⁷.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(16-18) alkyl group, or a C(16-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, n and m can be each independently from 3 to 6.

In some embodiments, examples of an ionizable molecule include compoundshaving the structure shown in Formula I

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, examples of an ionizable molecule include compoundshaving the structure shown in Formula I

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is selected from

whereineach R⁶ is independently hydroxyl;each R⁷ is independently selected from H, alkyl.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

Examples of an ionizable compound include the following compound A1:

which is((2-(3-(hydroxymethyl)azetidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate).

Examples of an ionizable compound include the following compound A2:

Examples of an ionizable compound include the following compound A3:

Examples of an ionizable compound include the following compound A4:

which is((2-((3S,4S)-3,4-dihydroxypyrrolidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate.

Examples of an ionizable compound include the following compound A5:

which is((2-((3R,4R)-3,4-dihydroxypyrrolidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate).

Examples of an ionizable compound include the following compound A6:

which is((2-((3S,4R)-3,4-dihydroxypyrrolidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate).

Examples of an ionizable compound include the following compound A7:

which is(((2-(dimethylamino)ethoxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate).

Examples of an ionizable compound include the following compound A8:

which is2-((bis(2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)ethyl)carbamoyl)oxy)-N,N,N-trimethylethan-1-aminium.

Examples of an ionizable compound include the following compound A9:

which is((2-(3-(hydroxymethyl)azetidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)ditetradecanoate.

Examples of an ionizable compound include the following compound AA:

which is((2-(4-(2-hydroxyethyl)piperazin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate).

Examples of an ionizable compound include the following compound AB:

which is((2-(4-(2-hydroxyethyl)piperazin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate).

Examples of an ionizable compound include the following compound AC:

Examples of an ionizable compound include the following compound AD:

Examples of an ionizable compound include the following compound AE:

Examples of an ionizable compound include the following compound AF:

Examples of an ionizable compound include the following compound B1:

Examples of an ionizable compound include the following compound B2:

Examples of an ionizable compound include the following compound B3:

Examples of an ionizable compound include the following compound B4:

Examples of an ionizable compound include the following compound B5:

Examples of an ionizable compound include the following compound B6:

Examples of an ionizable compound include the following compound B7:

Examples of an ionizable molecule include compounds having the structureshown in Formula II

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is a C(12-20) alkyl group or a C(12-20) alkenyl group that issubstituted with a carboxylic acid or ester group.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, R³ is a C(14-18) alkyl group, or a C(14-18) alkenylgroup that is substituted with a carboxylic acid or ester group.

Examples of an ionizable compound include the following compound B8:

Examples of an ionizable compound include the following compound B9:

Examples of an ionizable compound include the following compound BA:

Examples of an ionizable compound include the following compound BB:

In certain embodiments, examples of an ionizable compound includecompounds having the structure shown in Formula III

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is selected from alkyl, hydroxyalkyl, alkoxyalkoxy, andcarboxyalkyl;wherein R⁶ is selected from NR⁷ ₂, N⁺HR⁷ ₂ and N⁺R⁷ ₃;wherein R⁷ is selected from H, alkyl, hydroxyalkyl, and aminoalkyl.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(16-18) alkyl group, or a C(16-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

Examples of an ionizable compound include the following compound BC:

Examples of an ionizable compound include the following compound BD:

Examples of an ionizable compound include the following compound BE:

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

Examples of an ionizable compound include compounds having the structureshown in Formula IV

wherein R¹ and R² areR¹=C(═O)OR⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from 1-azetidines, 1-pyrrolidines, 1-piperidines,4-morpholines, and 1,4-piperazines wherein the rings can be substitutedat any carbon atom position,

and can also be selected from amino and aminoalkyl groups which can befurther substituted,

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;Q is O or NR⁷;p is from 1 to 4.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(16-18) alkyl group, or a C(16-18) alkenyl group.

In some embodiments, p is 1, 2, 3 or 4.

In some embodiments, q is 0, 1, 2, 3 or 4.

Examples of an ionizable compound include compounds having the structureshown in Formula IV

wherein R¹ and R² areR¹=C(═O)OR⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;p is from 1 to 4.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(16-18) alkyl group, or a C(16-18) alkenyl group.

In some embodiments, p is 1, 2, 3 or 4.

In some embodiments, q is 0, 1, 2, 3 or 4.

Examples of an ionizable compound include compounds having the structureshown in Formula IV

wherein R¹ and R² areR¹=C(═O)OR⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;p is from 1 to 4.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

Examples of an ionizable compound include compounds having the structureshown in Formula IV

wherein R¹ and R² areR¹=C(═O)OR⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

whereineach R⁶ is independently selected from H, hydroxyl, hydroxyalkyl,aminoalkyl;each R⁷ is independently selected from H, alkyl;p is from 1 to 4.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

Examples of an ionizable compound include the following compound BF:

Examples of an ionizable compound include the following compound C1:

Examples of an ionizable compound include the following compound C2:

which is2-((1-(((9,12Z)-heptadeca-9,12-dien-1-yl)oxy)-5-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)-1,5-dioxopentan-3-yl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium.

Examples of an ionizable compound include the following compound C3:

which is 1-((9Z,12Z)-heptadeca-9,12-dien-1-yl)5-((9Z,12Z)-octadeca-9,12-dien-1-yl)3-(2-(dimethylamino)acetamido)pentanedioate.

Examples of an ionizable compound include the following compound C4:

Examples of an ionizable compound include the following compound C5:

Examples of an ionizable compound include the following compound C6:

Examples of an ionizable compound include the following compound C7:

Examples of an ionizable compound include the following compound C8:

Examples of an ionizable compound include the following compound C9:

Examples of an ionizable compound include the following compound CA:

which is di((9Z,12Z)-octadeca-9,12-dien-1-yl)3-(2-(4-(2-hydroxyethyl)piperazin-1-yl)acetamido)pentanedioate.

Examples of an ionizable compound include the following compound CB:

Examples of an ionizable compound include the following compound CC:

Examples of an ionizable compound include the following compound CD:

Examples of an ionizable compound include the following compound CE:

Examples of an ionizable compound include the following compound CF:

Examples of an ionizable compound include the following compound D1:

which is di((9Z,12Z)-octadeca-9,12-dien-1-yl)3-(2-((3S,4R)-3,4-dihydroxypyrrolidin-1-yl)acetamido)pentanedioate.

Examples of an ionizable compound include the following compound D2:

Examples of an ionizable compound include the following compound D3:

Examples of an ionizable compound include the following compound D4:

Examples of an ionizable compound include the following compound D5:

Examples of an ionizable compound include the following compound D6:

Examples of an ionizable compound include the following compound D7:

Examples of an ionizable compound include compounds having the structureshown in Formula IV-B

wherein R¹ and R² areR¹=C(═O)OR⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein Z is S or O;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl;p is from 1 to 4.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(16-18) alkyl group, or a C(16-18) alkenyl group.

In some embodiments, p is 1, 2, 3 or 4.

Examples of an ionizable compound include the following compound D8:

Examples of an ionizable compound include the following compound D9:

Examples of an ionizable compound include the following compound DA:

Examples of an ionizable compound include the following compound DB:

Examples of an ionizable compound include the following compound DC:

Examples of an ionizable compound include the following compound DD:

which is di((9Z,12Z)-octadeca-9,12-dien-1-yl)3-((((2-(dimethylamino)ethyl)thio)carbonyl)amino)pentanedioate.

Examples of an ionizable compound include the following compound DE:

Examples of an ionizable compound include the following compound DF:

Examples of an ionizable compound include the following compound E1:

Examples of an ionizable compound include the following compound E2:

Examples of an ionizable compound include the following compound E3:

Examples of an ionizable compound include the following compound E4:

which is di((9Z,12Z)-octadeca-9,12-dien-1-yl)3-(((2-(dimethylamino)ethoxy)carbonyl)amino)pentanedioate.

Embodiments of this invention include compounds having the structureshown in Formula V

wherein R¹ and R² areR¹=NHC(═O)R⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein p is from 1 to 4;wherein R³ is selected from 1-azetidines, 1-pyrrolidines, 1-piperidines,4-morpholines, and 14-piperazines wherein the rings can be substitutedat any carbon atom position,

and can also be selected from amino and aminoalkyl groups which can besubstituted,

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;Q is O or NR⁷.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(16-18) alkyl group, or a C(16-18) alkenyl group.

In some embodiments, p is 1, 2, 3 or 4.

In some embodiments, q is 0, 1, 2, 3 or 4.

Embodiments of this invention include compounds having the structureshown in Formula V

wherein R¹ and R² areR¹=NHC(═O)R⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein p is from 1 to 4;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl;Q is O or NR⁷.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

Examples of an ionizable compound include the following compound E5:

Examples of an ionizable compound include the following compound E6:

Examples of an ionizable compound include the following compound E7:

Examples of an ionizable compound include the following compound E8:

Examples of an ionizable compound include the following compound E9:

Examples of an ionizable compound include the following compound EA:

Examples of an ionizable compound include the following compound EB:

Examples of an ionizable compound include the following compound EC:

Examples of an ionizable compound include the following compound ED:

Examples of an ionizable compound include the following compound EE:

Examples of an ionizable compound include the following compound EF:

Examples of an ionizable compound include the following compound F1:

Examples of an ionizable compound include the following compound F2:

Examples of an ionizable compound include the following compound F3:

Examples of an ionizable compound include the following compound F4:

Examples of an ionizable compound include compounds having the structureshown in Formula VI

wherein R¹ isR¹=OC(═O)R⁴wherein R² and R⁴ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from aminoalkyl, quaternary aminoalkyl.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R² and R⁴ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

Examples of an ionizable compound include the following compound F5:

which is2-((9Z,12Z)—N-(3-(dimethylamino)propyl)octadeca-9,12-dienamido)ethyl(9Z,12Z)-octadeca-9,12-dienoate.

Examples of an ionizable compound include the following compound F6:

which is2-((9Z,12Z)—N-(4-(dimethylamino)butyl)octadeca-9,12-dienamido)ethyl(9Z,12Z)-octadeca-9,12-dienoate.

Examples of an ionizable compound include the following compound F7:

which isN,N,N-trimethyl-3-((9Z,12Z)—N-(2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)ethyl)octadeca-9,12-dienamido)propan-1-aminium.

Examples of an ionizable compound include the following compound F8:

which is 2-(N-(3-(dimethylamino)propyl)tetradecanamido)ethyltetradecanoate.

Examples of an ionizable compound include the following compound F9:

which isN,N,N-trimethyl-3-(N-(2-(tetradecanoyloxy)ethyl)tetradecanamido)propan-1-aminium.

Examples of an ionizable compound include compounds having the structureshown in Formula VII

wherein R¹ and R² areR¹=OC(═O)R⁴R²=OC(═O)R⁵wherein n and m are each independently from 1 to 2; and R⁴ and R⁵ areindependently for each occurrence a C(12-20) alkyl group, or a C(12-20)alkenyl group;wherein R³ is selected from H, alkyl, aminoalkyl, quaternary aminoalkyl,hydroxyalkyl, alkoxyalkyl, alkoxyalkoxyalkyl.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

Examples of an ionizable compound include the following compound FA:

Examples of an ionizable compound include the following compound FB:

Examples of an ionizable compound include the following compound FC:

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In certain embodiments, a compound can have the structure shown inFormula VIII

wherein R¹ and R² areR¹=OC(═O)R⁴R²=C(═O)ZR⁵wherein Z is NH or O,wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

amino;

quaternary amino;

aminoalkyl;

quaternary aminoalkyl;

NHC(═O)(CH₂)R¹⁰;NHC(═O)SR⁹;wherein R¹⁰ is selected from 1-azetidines, 1-pyrrolidines,1-piperidines, 4-morpholines, and 1,4-piperazines wherein the rings canbe substituted at any carbon atom position,

and can also be selected from amino and aminoalkyl groups which can besubstituted

carboxyalkyl;

aminoalkyl;

wherein R⁹ is selected from

alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

and wherein

each R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;

each R⁷ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl;

each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;

q is from zero to four;

p is from 1 to 4;

Q is or NR⁷.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(16-18) alkyl group, or a C(16-18) alkenyl group.

In some embodiments, p is 1, 2, 3 or 4.

In some embodiments, q is 0, 1, 2, 3 or 4.

In certain embodiments, a compound can have the structure shown inFormula VIII

wherein R¹ and R² areR¹=OC(═O)R⁴R²=C(═O)ZR⁵wherein Z is NH or O,wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

NHC(═O)(CH₂)_(p)R¹⁰;

NHC(═O)SR⁹;

wherein R¹⁰ is selected from

carboxyalkyl

aminoalkyl

wherein R⁹ is selected from

alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

and wherein

each R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;

each R⁷ is independently selected from H, alkyl, hydroxyalkyl;

each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;

p is from 1 to 4;

q is from zero to four;

Q is O or NR⁷.

In certain embodiments, a compound can have the structure shown inFormula VIII

wherein R¹ and R² areR¹=OC(═O)R⁴R²=C(═O)ZR⁵wherein Z is NH or O,wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

NHC(═O)(CH₂)_(p)R¹⁰;

NHC(═O)SR⁹;

wherein R¹⁰ is selected from

carboxyalkyl;

aminoalkyl;

wherein R⁹ is selected from

alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

and wherein

each R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;

each R⁷ is independently selected from H, alkyl, hydroxyalkyl;

each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;

p is from 1 to 4;

q is from zero to four;

Q is or NR⁷.

In certain embodiments, a compound can have the structure shown inFormula VIII

wherein R¹ and R² areR¹=OC(═O)R⁴R²=C(═O)ZR⁵wherein Z is NH or O,wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected fromNHC(═O)(CH₂)_(p)R¹⁰NHC(═O)SR⁹;wherein R¹⁰ is selected from

wherein R⁹ is selected from

alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

and wherein

each R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;

each R⁷ is independently selected from H, alkyl, hydroxyalkyl;

Q is or NR⁷.

In certain embodiments, a compound can have the structure shown inFormula VIII

wherein R¹ and R² areR¹=OC(═O)R⁴R²=C(═O)ZR⁵wherein Z is NH,wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

NHC(═O)(CH₂)_(p)R¹⁰;

NHC(═O)SR⁹;

wherein R¹⁰ is selected from

wherein R⁹ is selected from

alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl; and wherein

each R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;

each R⁷ is independently selected from H, alkyl, hydroxyalkyl;

Q is or NR⁷.

Examples of an ionizable compound include the following compound FD:

Examples of an ionizable compound include the following compound FE:

Examples of an ionizable compound include the following compound FF:

Examples of an ionizable compound include the following compound A11:

Examples of an ionizable compound include the following compound A12:

Examples of an ionizable compound include the following compound A13:

Examples of an ionizable compound include the following compound A14:

Examples of an ionizable compound include the following compound A15:

Examples of an ionizable compound include the following compound A16:

Examples of an ionizable compound include the following compound A17:

Examples of an ionizable compound include the following compound A18:

Examples of an ionizable compound include the following compound A19:

Examples of an ionizable compound include the following compound A20:

Examples of an ionizable compound include the following compound A21:

Examples of an ionizable compound include the following compound A22:

Examples of an ionizable compound include the following compound A23:

which is(S)-4-oxo-4-((1-oxo-3-(tetradecanoyloxy)-1-(tetradecylamino)propan-2-yl)amino)butanoicacid.

Examples of an ionizable compound include the following compound A24

which is(S)-2-(2-(4-(3-hydroxypropyl)piperazin-1-yl)acetamido)-3-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)-3-oxopropyl(9Z,12Z)-octadeca-9,12-dienoate.

Examples of an ionizable compound include the following compound A25:

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In further embodiments, a compound can have the structure shown inFormula VIII-B

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵wherein n and m are each independently from 1 to 2;R⁴ and R⁵ are independently for each occurrence a C(12-20) alkyl group,or a C(12-20) alkenyl group;wherein Z is N, O;wherein R³ is selected fromalkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl;p is from 1 to 4.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

Examples of an ionizable compound include the following compound B11:

Examples of an ionizable compound include the following compound B12:

Examples of an ionizable compound include the following compound B13:

Examples of an ionizable compound include the following compound B14:

In additional embodiments, a compound can have the structure shown inFormula IX

wherein R¹ and R² areR¹=C(═O)OR⁴R²=NHC(═O)R⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein p is from 1 to 4;wherein R³ is selected from 1-azetidines, 1-pyrrolidines, 1-piperidines,4-morpholines, and 1,4-piperazines wherein the rings can be substitutedat any carbon atom position,

and can also be selected from amino and aminoalkyl groups which can besubstituted,

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;Q is O or NR⁷.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(16-18) alkyl group, or a C(16-18) alkenyl group.

In some embodiments, p is 1, 2, 3 or 4.

In some embodiments, q is 0, 1, 2, 3 or 4.

In additional embodiments, a compound can have the structure shown inFormula IX

wherein R¹ and R² areR¹=C(═O)OR⁴R²=NHC(═O)R⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein p is from 1 to 4;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In additional embodiments, a compound can have the structure shown inFormula IX

wherein R¹ and R² areR¹=C(═O)OR⁴R²=NHC(═O)R⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein p is from 1 to 4;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

Examples of an ionizable compound include the following compound C11:

Examples of an ionizable compound include the following compound C12:

Examples of an ionizable compound include the following compound C13:

Examples of an ionizable compound include the following compound C14:

Examples of an ionizable compound include the following compound C15:

Examples of an ionizable compound include the following compound C16:

Examples of an ionizable compound include the following compound C127

Examples of an ionizable compound include the following compound C18:

Examples of an ionizable compound include the following compound C19:

Examples of an ionizable compound include the following compound C20:

Examples of an ionizable compound include the following compound C21:

Examples of an ionizable compound include the following compound C22:

Examples of an ionizable compound include the following compound C23:

Examples of an ionizable compound include the following compound C24:

which isN,N,N-trimethyl-2-(((S)-3-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)-2-((9Z,12Z)-octadeca-9,12-dienamido)-3-oxopropyl)amino)-2-oxoethan-1-aminium.

Examples of an ionizable compound include the following compound C25:

which is (9Z,12Z)-octadeca-9,12-dien-1-yl(S)-3-(2-(dimethylamino)acetamido)-2-((9Z,12Z)-octadeca-9,12-dienamido)propanoate.

In some embodiments, a compound can have the structure shown in FormulaX

wherein R¹ and R² areR¹=C(═O)OR⁴R²=NHC(═O)R⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein R³ is selected from

amino;

quaternary amino;

aminoalkyl;

quaternary aminoalkyl;

hydroxyalkylamino.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

Examples of an ionizable compound include the following compound D11:

Examples of an ionizable compound include the following compound D12:

Examples of an ionizable compound include the following compound D13:

Examples of an ionizable compound include the following compound D14:

Embodiments of this invention include compounds having the structureshown in Formula XI

wherein R¹ and R² areR¹=C(═O)R⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein Z is O or NH;wherein p is from 1 to 4;wherein R³ is selected from 1-azetidines, 1-pyrrolidines, 1-piperidines,4-morpholines, and 1,4-piperazines wherein the rings can be substitutedat any carbon atom position,

and can also be selected from amino and aminoalkyl groups which can besubstituted,

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four;Q is O or NR⁷.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(16-18) alkyl group, or a C(16-18) alkenyl group.

In some embodiments, p is 1, 2, 3 or 4.

In some embodiments, q is 0, 1, 2, 3 or 4.

Embodiments of this invention include compounds having the structureshown in Formula XI

wherein R and R areR¹=C(═O)R⁴R²=C(═O)OR⁵wherein R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group;wherein Z is O or NH;wherein p is from 1 to 4;wherein R³ is selected from

whereineach R⁶ is independently selected from H, alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkoxy, aminoalkyl;each R⁷ is independently selected from H, alkyl;each R⁸ is independently selected from H, alkyl, hydroxyalkyl, andaminoalkyl, and any two R⁸ may form a ring;q is from zero to four.

In some embodiments, each of the alkenyl groups can have from one to twodouble bonds.

In some embodiments, R⁴ and R⁵ are independently for each occurrence aC(14-18) alkyl group, or a C(14-18) alkenyl group.

In some embodiments, the terms alkyl, hydroxyalkyl, and aminoalkyl referto C(1-6)alkyl, hydroxyl[C(1-6)alkyl], and amino[C(1-6)alkyl].

Examples of an ionizable compound include the following compound E11:

Examples of an ionizable compound include the following compound E12:

Examples of an ionizable compound include the following compound E13:

Examples of an ionizable compound include the following compound E14

Examples of an ionizable compound include the following compound E15:

Examples of an ionizable compound include the following compound E16:

Examples of an ionizable compound include the following compound E17:

Examples of an ionizable compound include the following compound E18:

Examples of an ionizable compound include the following compound E19:

Examples of an ionizable compound include the following compound E20:

Examples of an ionizable compound include the following compound E21:

Examples of an ionizable compound include the following compound E22:

Examples of an ionizable compound include the following compound E23:

Examples of an ionizable compound include the following compound E24:

Examples of an ionizable compound include the following compound E25:

Examples of an ionizable compound include the following compound E26:

Examples of an ionizable compound include the following compound E27:

Examples of an ionizable compound include the following compound E28:

Examples of an ionizable compound include the following compound E29:

Examples of an ionizable compound include the following compound E30:

Examples of an ionizable compound include the following compound E31:

Examples of an ionizable compound include the following compound E32:

Examples of an ionizable compound include the following compound E33:

Examples of an ionizable compound include the following compound E34:

Examples of an ionizable compound include the following compound E35:

Examples of an ionizable compound include the following compound E36:

Examples of an ionizable compound include the following compound E37:

which is (9Z,12Z)-octadeca-9,12-dien-1-yl(2S,4R)-4-(3-(dimethylamino)propanamido)-1-((9Z,12Z)-octadeca-9,12-dienoyl)pyrrolidine-2-carboxylate.

Examples of an ionizable compound include the following compound E38

which is (9Z,12Z)-octadeca-9,12-dien-1-yl(2S,4R)-4-(4-(dimethylamino)butanamido)-1-((9Z,12Z)-octadeca-9,12-dienoyl)pyrrolidine-2-carboxylate.

Examples of an ionizable compound include the following compound E39:

which isN,N,N-trimethyl-4-(((3R,5S)-5-((((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)carbonyl)-1-((9Z,12Z)-octadeca-9,12-dienoyl)pyrrolidin-3-yl)amino)-4-oxobutan-1-aminium.

Examples of an ionizable compound include the following compound E40:

Structural Lipids

Examples of structural lipids include cholesterols, sterols, andsteroids.

Examples of structural lipids include cholanes, cholestanes, ergostanes,campestanes, poriferastanes, stigmastanes, gorgostanes, lanostanes,gonanes, estranes, androstanes, pregnanes, and cycloartanes.

Examples of structural lipids include sterols and zoosterols such ascholesterol, lanosterol, zymosterol, zymostenol, desmosterol,stigmastanol, dihydrolanosterol, and 7-dehydrocholesterol.

Examples of structural lipids include pegylated cholesterols, andcholestane 3-oxo-(C1-22)acyl compounds, for example, cholesterylacetate, cholesteryl arachidonate, cholesteryl butyrate, cholesterylhexanoate, cholesteryl myristate, cholesteryl palmitate, cholesterylbehenate, cholesteryl stearate, cholesteryl caprylate, cholesteryln-decanoate, cholesteryl dodecanoate, cholesteryl nervonate, cholesterylpelargonate, cholesteryl n-valerate, cholesteryl oleate, cholesterylelaidate, cholesteryl erucate, cholesteryl heptanoate, cholesteryllinolelaidate, and cholesteryl linoleate.

Examples of structural lipids include sterols such as phytosterols,beta-sitosterol, campesterol, ergosterol, brassicasterol,delta-7-stigmasterol, and delta-7-avenasterol.

Stabilizer Lipids

Examples of stabilizer lipids include zwitterionic lipids.

Examples of stabilizer lipids include compounds such as phospholipids.

Examples of phospholipids include phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine and ordilinoleoylphosphatidylcholine.

Examples of stabilizer lipids include phosphatidyl ethanolaminecompounds and phosphatidyl choline compounds.

Examples of stabilizer lipids include1,2-dioleoyl-sn-Glycero-3-Phosphocholine (DOPC).

Examples of stabilizer lipids include diphytanoyl phosphatidylethanolamine (DPhPE) and 1,2-Diphytanoyl-sn-Glycero-3-Phosphocholine(DPhPC).

Examples of stabilizer lipids include1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

Examples of stabilizer lipids include 1,2-dilauroyl-sn-glycerol (DLG);1,2-dimyristoyl-sn-glycerol (DMG); 1,2-dipalmitoyl-sn-glycerol (DPG);1,2-distearoyl-sn-glycerol (DSG);1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC);1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC);1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);1,2-dipalmitoyl-sn-glycero-O-ethyl-3-phosphocholine (DPePC);1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE);1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE);1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE);1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (P-Lyso-PC); and1-Stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-Lyso-PC).

Lipids for Reducing Immunogenicity

Examples of lipids for reducing immunogenicity include polymericcompounds and polymer-lipid conjugates.

Examples of lipids for reducing immunogenicity include pegylated lipidshaving polyethyleneglycol (PEG) regions. The PEG regions can be of anymolecular mass. In some embodiments, a PEG region can have a molecularmass of 200, 300, 350, 400, 500, 550, 750, 1000, 1500, 2000, 3000, 3500,4000 or 5000 Da.

Examples of lipids for reducing immunogenicity include compounds havinga methoxypolyethyleneglycol region.

Examples of lipids for reducing immunogenicity include compounds havinga carbonyl-methoxypolyethyleneglycol region.

Examples of lipids for reducing immunogenicity include compounds havinga multi-branched PEG region.

Examples of lipids for reducing immunogenicity include compounds havinga polyglycerine region.

Examples of lipids for reducing immunogenicity include polymeric lipidssuch as DSPE-mPEG, DMPE-mPEG, DPPE-mPEG, and DOPE-mPEG.

Examples of lipids for reducing immunogenicity include PEG-phospholipidsand PEG-ceramides.

Cationic Lipids

Examples of cationic lipids include cationic HEDC compounds as describedin US 2013/0330401 A1. Some examples of cationic lipids are given in US2013/0115274 A1.

Lipid Compositions

In some embodiments, a composition can contain the ionizable compoundA6, the structural lipid cholesterol, the stabilizer lipids DOPC andDOPE, and the lipid for reducing immunogenicity DPPE-mPEG. In certainembodiments, compound A6 can be 15 to 25 mol % of the composition; thecholesterol, DOPC, and DOPE combined can be 75 to 85 mol % of thecomposition; and DPPE-mPEG can be 5 mol % of the composition.

In one embodiment, compound A6 can be 25 mol % of the composition;cholesterol can be 30 mol % of the composition, DOPC can be 20 mol % ofthe composition, DOPE can be 20 mol % of the composition; andDPPE-mPEG(2000) can be 5 mol % of the composition.

Nanoparticles

Embodiments of this invention can provide liposome nanoparticlecompositions. The ionizable molecules of this invention can be used toform liposome compositions, which can have one or more bilayerstructures of lipid-like molecules.

A nanoparticle composition can have one or more of the ionizablemolecules of this invention in a liposomal structure, a bilayerstructure, a micelle, a lamellar structure, or a mixture thereof.

In some embodiments, a composition can include one or more liquidvehicle components. A liquid vehicle suitable for delivery of activeagents of this invention can be a pharmaceutically acceptable liquidvehicle. A liquid vehicle can include an organic solvent, or acombination of water and an organic solvent.

Embodiments of this invention can provide lipid nanoparticles having asize of from 10 to 1000 nm. In some embodiments, the liposomenanoparticles can have a size of from 10 to 150 nm.

Pharmaceutical Compositions

This invention further contemplates methods for distributing an activeagent to an organ of a subject for treating fibrosis by administering tothe subject a composition of this invention. Organs that can be treatedinclude lung, liver, pancreas, kidney, colon, heart, bone marrow, skin,intestine, brain and eye.

In some embodiments, this invention provides methods for treating a lungfibrosis disease by administering to the subject a composition of thisinvention.

Examples of fibrosis disease include idiopathic lung fibrosis and livercirrhosis.

In further aspects, this invention provides a range of pharmaceuticalformulations.

A pharmaceutical formulation herein can include an active agent, as wellas a drug carrier, or a lipid of this invention, along with apharmaceutically acceptable carrier or diluent. In general, activeagents of this description include siRNAs, active agents for fibrosis,as well as any small molecule drug.

A pharmaceutical formulation of this invention may contain one or moreof each of the following: a surface active agent, a diluent, anexcipient, a preservative, a stabilizer, a dye, and a suspension agent.

Some pharmaceutical carriers, diluents and components for apharmaceutical formulation, as well as methods for formulating andadministering the compounds and compositions of this invention aredescribed in Remington's Pharmaceutical Sciences, 18th Ed., MackPublishing Co., Easton, Pa. (1990).

Examples of preservatives include sodium benzoate, ascorbic acid, andesters of p-hydroxybenzoic acid.

Examples of surface active agents include alcohols, esters, sulfatedaliphatic alcohols.

Examples of excipients include sucrose, glucose, lactose, starch,crystallized cellulose, mannitol, light anhydrous silicate, magnesiumaluminate, magnesium metasilicate aluminate, synthetic aluminumsilicate, calcium carbonate, sodium acid carbonate, calcium hydrogenphosphate, and calcium carboxymethyl cellulose.

Examples of suspension agents include coconut oil, olive oil, sesameoil, peanut oil, soya, cellulose acetate phthalate,methylacetate-methacrylate copolymer, and ester phthalates.

Structures of Molecular Tails

A compound of this invention may have one or more lipophilic tails thatcontain one or more alkyl or alkenyl groups. Examples of lipophilictails having alkenyl groups include C(14:1(5))alkenyl,C(14:1(9))alkenyl, C(16:1(7))alkenyl, C(16:1(9))alkenyl,C(18:1(3))alkenyl, C(18:1(5))alkenyl, C(18:1(7))alkenyl,C(18:1(9))alkenyl, C(18:1(11))alkenyl, C(18:1(12))alkenyl,C(18:2(9,12))alkenyl, C(18:2(9,11))alkenyl, C(18:3(9,12,15))alkenyl,C(18:3(6,9,12))alkenyl, C(18:3(9,11,13))alkenyl,C(18:4(6,9,12,15))alkenyl, C(18:4(9,11,13,15))alkenyl,C(20:1(9))alkenyl, C(20:1(11))alkenyl, C(20:2(8,11))alkenyl,C(20:2(5,8))alkenyl, C(20:2(11,14))alkenyl, C(20:3(5,8,11))alkenyl,C(20:4(5,8,11,14))alkenyl, C(20:4(7,10,13,16))alkenyl,C(20:5(5,8,11,14,17))alkenyl, C(20:6(4,7,10,13,16,19))alkenyl,C(22:1(9))alkenyl, C(22:1(13))alkenyl, and C(24:1(9))alkenyl. Someexamples of tail structures are found at Donald Voet and Judith Voet,Biochemistry, 3rd Edition (2005), p. 383.

Some examples of lipophilic tails include the following structures:

Any of these example structures of lipophilic tails may have one or moreadditional chemical branches.

Chemical Definitions

The term “alkyl” as used herein refers to a hydrocarbyl radical of asaturated aliphatic group, which can be of any length. An alkyl groupcan be a branched or unbranched, substituted or unsubstituted aliphaticgroup containing from 1 to 22 carbon atoms. This definition also appliesto the alkyl portion of other groups such as, for example, cycloalkyl,alkoxy, alkanoyl, and aralkyl, for example.

As used herein, for example, a term such as “C(1-5)alkyl” includesC(1)alkyl, C(2)alkyl, C(3)alkyl, C(4)alkyl, and C(5)alkyl. Likewise, forexample, the term “C(3-22)alkyl” includes C(1)alkyl, C(2)alkyl,C(3)alkyl, C(4)alkyl, C(5)alkyl, C(6)alkyl, C(7)alkyl, C(8)alkyl,C(9)alkyl, C(10)alkyl, C(11)alkyl, C(12)alkyl, C(13)alkyl, C(14)alkyl,C(15)alkyl, C(16)alkyl, C(17)alkyl, C(18)alkyl, C(19)alkyl, C(20)alkyl,C(21)alkyl, and C(22)alkyl.

As used herein, an alkyl group may be designated by a term such as Me(methyl, —CH₃), Et (ethyl, —CH₂CH₃), Pr (any propyl group), ^(n)Pr(n-Pr, n-propyl), ^(i)Pr (i-Pr, isopropyl), Bu (any butyl group), ^(n)Bu(n-Bu, n-butyl), ^(i)Bu (i-Bu, isobutyl), ^(s)Bu (s-Bu, sec-butyl), and^(t)Bu (t-Bu, tert-butyl).

The term “alkenyl” as used herein refers to hydrocarbyl radical havingat least one carbon-carbon double bond. An alkenyl group can be branchedor unbranched, substituted or unsubstituted hydrocarbyl radical having 2to 22 carbon atoms and at least one carbon-carbon double bond. An“alkenyl” group has one or more carbon-carbon double bonds.

The term “substituted” as used herein refers to an atom having one ormore substitutions or substituents which can be the same or differentand may include a hydrogen substituent. Thus, the terms alkyl,cycloalkyl, alkenyl, alkoxy, alkanoyl, and aryl, for example, refer togroups which can include substituted variations. Substituted variationsinclude linear, branched, and cyclic variations, and groups having asubstituent or substituents replacing one or more hydrogens attached toany carbon atom of the group.

In general, a compound may contain one or more chiral centers. Compoundscontaining one or more chiral centers may include those described as an“isomer,” a “stereoisomer,” a “diastereomer,” an “enantiomer,” an“optical isomer,” or as a “racemic mixture.” Conventions forstereochemical nomenclature, for example the stereoisomer naming rulesof Cahn, Ingold and Prelog, as well as methods for the determination ofstereochemistry and the separation of stereoisomers are known in theart. See, for example, Michael B. Smith and Jerry March, March'sAdvanced Organic Chemistry, 5th edition, 2001. The compounds andstructures of this disclosure are meant to encompass all possibleisomers, stereoisomers, diastereomers, enantiomers, and/or opticalisomers that would be understood to exist for the specified compound orstructure, including any mixture, racemic or otherwise, thereof.

This invention encompasses any and all tautomeric, solvated orunsolvated, hydrated or unhydrated forms, as well as any atom isotopeforms of the compounds and compositions disclosed herein.

This invention encompasses any and all crystalline polymorphs ordifferent crystalline forms of the compounds and compositions disclosedherein.

Abbreviations Used

DMAP—4-N,N-Dimethylaminopyridine

DCM—Dichloromethane

TEA—Triethylamine

ED—1-(3-Dimethylaminopropyl)-3-ethylcarbodimimde hydrochloride

Na₂SO₄—Sodium sulphate

EtOAc—Ethyl acetate

DMF—N,N-Dimethylformide

ELSD—Evaporating Light Scattering Detector

NaCl—Sodium chloride

K₂CO₃—Potassium carbonate

MeOH—Methanol

TFA—Trifluoroacetic acid

DIEA—N,N-Diisopropylethylamine

MgSO₄—Magnesium sulphate

LCMS—Liquid chromatography-mass spectrometry

NaHCO₃—Sodium bicarbonate

H₂O—Water

HCl—Hydrochloride

KI—Potassium idoide

DMSO—Dimethyl sulfoxide

TBAF—tetra-N-Butylammonium fluoride

NaBH₄—Sodium borohydride

THF—Tetrahydrofuran

TBDMS—tert-Butyldimethylsilyl

LiOH—Lithium hydroxide

MeI—Methyl iodide

BOC—tert-Butyloxycarbonyl

Fmoc—Fluorenylmethyloxycarbonyl

EXAMPLES Example 1

A scheme for the preparation of Compound A6 is shown in FIG. 1.

Intermediate 1: Linoleic acid (50.0 g, 178.30 mmol),N-Boc-diethanolamine (18.3 g, 89.10 mmol), and DMAP (1.1 g, 8.90 mmol)in an oven-dried flask (1 L) with a magnetic bar was added anhydrous DCM(400 mL). The mixture was stirred at ambient temperature for 2 minutesto a clear solution. EDC (35.9 g, 187.20 mmol) was then added and themixture was stirred at room temperature overnight (17 hours). Thereaction was finally quenched with saturated NaCl aqueous solution (400mL) and extracted with DCM twice (400 mL, 100 mL). Organic layers werecombined, dried over Na₂SO₄ (20 g), and filtered. The filtrate wasconcentrated under reduced pressure. The crude was dissolved in 50 mLDCM and purified by flash chromatography purification system (330 gsilica gel column) using a gradient of hexane for 5 min, then 0-20%EtOAc/hexane for 40 min under the flow rate at 100 mL/min. The productfractions were collected and concentrated to yield Intermediate 1 (59 g,91% yield) as a clear liquid. ¹H nmr (400 MHz, CDCl₃) δ: 5.32-5.33 (8H,m, CH═), 4.13-4.17 (4H, m, OCH₂), 3.43-3.49 (4H, m, NCH₂), 2.73-2.74(4H, m, ═CHCH₂CH═), 2.03-2.28 (4H, m, CH₂CO), 2.00-2.01 (8H, m, ═CHCH₂),1.60-1.70 (4H, m, CH₂CH₂CO), 1.43 (9H, S, C(CH₃)₃), 1.28-1.31 (28H, m,CH₂), 0.85-0.86 (6H, m, CH₃).

Intermediate 2: Intermediate 1 (50.0 g, 68.50 mmol) in an oven-driedflask (1000 mL) with a magnetic bar was added anhydrous DCM (100 mL).The mixture was stirred for 2 minutes to a clear solution. TFA (100 mL)was then added and the mixture was stirred at ambient temperature for 2hours. During stirring, the solution color turned from clear to red.Next, the reaction mixture was first concentrated using a rotavapor andwashed with saturated NaHCO₃ aqueous solution (300 mL). The mixture wasthen extracted with DCM (2×200 mL) and the organic layers were combined,dried over Na₂SO₄ (20 g), filtered, and concentrated under reducedpressure to give an oily intermediate, which was then dissolved inanhydrous DCM (300 mL) and cooled down to 0° C. with an ice-water bath.Chloroacetyl chloride (6.0 mL, 75.30 mmol) followed by DIEA (14.3 mL,82.20 mmol) were then added slowly at 0° C. After the addition wascompleted, the ice-water bath was removed and the mixture was stirred atambient temperature for 2 hours. Next, the reaction mixture wasconcentrated using a rotavapor, washed with saturated NaHCO₃ aqueoussolution (300 mL), and extracted with DCM (300 mL, 200 mL). The combinedorganic layers were then dried over Na₂SO₄ (20 g), filtered, andconcentrated under reduced pressure. The crude was dissolved in 20 mLDCM and purified with a 330 g silica column using a gradient of hexanefor 5 min, then 10% EtOAc/hexane for 20 min followed by 20% EtOAc/hexanefor 20 min under the flow rate at 60 mL/min. The product fractions werecollected and concentrated to yield Intermediate 2 (41.4 g, 82% yield)as a clear yellow liquid. ¹H nmr (400 MHz, CDCl₃) δ: 5.35-5.33 (8H, m,CH═), 4.24-4.22 (4H, m, OCH₂),4.14 (2H, s, CH₂Cl), 3.67-3.63 (4H, m,NCH₂), 2.78-2.75 (4H, m, ═CHCH₂CH═), 2.30-2.29 (4H, m, CH₂CO), 2.03-2.05(8H, m, ═CHCH₂), 1.60-1.56 (4H, m, CH₂CH₂CO), 1.33-1.29 (28H, m, CH₂),0.90-0.86 (6H, m, CH₃).

Compound A6: Intermediate 2 (5.0 g, 7.07 mmol), KI (1.2 g, 7.07 mmol)and cis-3,4-dihydroxy-pyrrolidine hydrochloride (1.3 g, 9.20 mmol) in anoven-dried vial (200 mL) with a magnetic bar was added anhydrous DMF (20mL). The mixture was stirred at ambient temperature for 2 minutes to aclear solution. DIEA (3.0 mL, 9.91 mmol) was then added and the mixturewas stirred at 30° C. for 2.5 hours. After removed the solvent byrotavapor under high vacuum, the residue was added with saturated NaHCO₃solution (100 mL) and extracted with DCM (100 mL, 50 mL). The organiclayers were then combined, dried over Na₂SO₄ (20 g), filtered, andconcentrated under reduced pressure. The crude was dissolved in 5 mL DCMand purified with a 120 g silica column using a gradient of hexane for 5min, then 0-50% EtOAc/hexane for 10 min followed by 50% EtOAc/hexane for10 min, then with DCM for 5 min and followed by 5% MeOH/DCM for 30 minunder the flow rate at 60 mL/min. The product fractions were collectedand concentrated to yield Compound A6 (3.5 g, 64% yield) as a yellowliquid. ¹H nmr (400 MHz, CDCl₃) δ: 5.34-5.29 (8H, m, CH═), 4.22-4.20(6H, m, OCH₂, OCH), 3.59-3.49 (6H, m, NCH₂, COCH₂N), 2.95-2.74 (6H, m,═CHCH₂CH═, NCH₂), 2.30-2.29 (4H, m, CH₂CO), 2.05-2.03 (8H, m, ═CHCH₂),1.60 (4H, m, CH₂CH₂CO), 1.33-1.29 (28H, m, CH₂), 0.90-0.88 (6H, m, CH₃).

Example 2

A scheme for the preparation of Compound AB is shown in FIG. 2.

Intermediate 3: Intermediate 1 (10.0 g, 13.70 mmol) in an oven-driedflask (200 mL) with a magnetic bar was added anhydrous DCM (15 mL). Themixture was stirred for 2 minutes to a clear solution. TFA (15 mL) wasthen added and the mixture was stirred at ambient temperature for 2hours. During stirring, the solution color turned from clear to red.Next, the reaction mixture was first concentrated using a rotavapor.Residue was diluted with DCM (˜50 mL) and 10% K₂CO₃ (15 mL) was addedand stirred for 10-15 minutes in an ice bath. pH of aqueous layer waschecked to ensure pH>8. Mixture was then transferred to a separatoryfunnel & extracting with DCM (50 mL, 25 mL), dried over MgSO₄, filtered,and concentrated under reduced pressure to give an oily intermediate,which was then dissolved in anhydrous DCM (30 mL) and bromoacetylbromide (1.2 mL, 13.70 mmol) followed by TEA (2.1 mL, 15.07 mmol) werethen added slowly. After the addition was completed, the mixture wasstirred at ambient temperature overnight. The reaction mixture was thenconcentrated using a rotavapor, washed with water (30 mL), and extractedwith DCM (30 mL, 20 mL). The combined organic layers were then driedover MgSO₄, filtered, and concentrated under reduced pressure. The crudewas dissolved in 20 mL DCM and purified with a 330 g silica column usinga gradient of hexane for 0.5 min, then 0-20% EtOAc/hexane 30 mingradient followed by 30% EtOAc/hexane for 5 min under the flow rate at130 mL/min. The product fractions were collected and concentrated toyield Intermediate 3 (8.1 g, 79% yield) as a clear yellow liquid. ¹H nmr(400 MHz, CDCl₃) δ: 5.35-5.33 (8H, m, CH═), 4.24-4.22 (4H, m, OCH₂),3.93 (2H, s, CH₂Br), 3.69-3.61 (4H, m, NCH₂), 2.78-2.74 (4H, m,═CHCH₂CH═), 2.32-2.28 (4H, m, CH₂CO), 2.06-2.02 (8H, m, ═CHCH₂),1.62-1.60 (4H, m, CH₂CH₂CO), 1.36-1.26 (28H, m, CH₂), 0.90-0.86 (6H, m,CH₃).

Compound AB: Intermediate 3 (460 mg, 0.61 mmol) and1-piperazine-propanol (89 mg, 0.61 mmol) in an oven-dried vial (100 mL)with a magnetic bar was added anhydrous DCM (10 mL). The mixture wasstirred at ambient temperature for 2 minutes to a clear solution. TEA(103 μL, 0.74 mmol) was then added and the mixture was stirred atambient temperature overnight. After removed the solvent by a rotavaporunder high vacuum, the residue was added with saturated 10% K₂CO₃solution (50 mL) and extracted with DCM (50 mL, 25 mL). The organiclayers were then combined, dried over MgSO₄, filtered, and concentratedunder reduced pressure. The crude was purified by flash chromatographypurification system. Material directly loaded onto a 24 g silica columnand using a gradient of DCM for 3 min, then 0-8% MeOH/DCM for 7 minfollowed by 8% MeOH/DCM for 20 min under the flow rate at 25 mL/min. Theproduct fractions were collected and concentrated to yield Compound AB(379 mg, 76% yield) as a yellow liquid. ¹H nmr (400 MHz, CDCl₃) δ:5.39-5.29 (8H, m, CH═), 4.22-4.21 (4H, m, OCH₂), 3.74-3.71 (2H, m,CH₂OH), 3.59-3.49 (4H, m, NCH₂), 3.23 (2H, s, COCH₂), 2.78-2.75 (2H, m,CH₂CH₂OH), 2.59-2.57 (8H, m, NCH₂CH₂N), 2.32-2.27 (4H, m, CHCH₂CH),2.05-2.03 (8H, m, ═CHCH₂CH₂), 1.60 (4H, m, CH₂CH₂CO), 1.33-1.29 (28H, m,CH₂), 0.90-0.88 (6H, m, CH₃).

Example 3

A scheme for the preparation of Compound A4 is shown in FIG. 3.

Intermediate 4: Myristic acid (50.0 g, 218.90 mmol),N-Boc-diethanolamine (21.4 g, 218.90 mmol), and DMAP (3.8 g, 65.70 mmol)in an oven-dried flask (1 L) with a magnetic bar was added anhydrous DCM(300 mL). The mixture was stirred at ambient temperature for 2 minutesto a clear solution. EDC (44.0 g, 481.70 mmol) was then added and themixture was stirred at room temperature overnight (17 hours). Thereaction was finally quenched with saturated NaCl solution (400 mL) andextracted with DCM twice (400 mL, 100 mL). Organic layers were combined,dried over Na₂SO₄ (20 g), and filtered. The filtrate was concentratedunder reduced pressure. The crude was dissolved in 50 mL DCM andpurified by flash chromatography purification system (330 g silica gelcolumn) using a gradient of 5-50% EtOAc/hexane for 40 min under the flowrate at 100 mL/min. The product fractions were collected andconcentrated to yield Intermediate 4 (50.0 g, 71% yield) as a whitesolid. ¹H nmr (400 MHz, CDCl₃) δ: 4.14-4.17 (4H, m, OCH₂), 3.44-3.50(4H, m, NCH₂), 2.27-2.30 (4H, m, CH₂CO), 1.60-1.70 (4H, m, CH₂CH₂CO),1.45 (9H, S, C(CH₃)₃), 1.20-1.25 (40H, m, CH₂), 0.81-0.88 (6H, m, CH₃).

Intermediate 5: Intermediate 4 (10.0 g, 16.00 mmol) in an oven-driedflask (200 mL) with a magnetic bar was added anhydrous DCM (15 mL). Themixture was stirred for 2 minutes to a clear solution. TFA (15.0 mL) wasthen added and the mixture was stirred at ambient temperature for 2hours. The reaction mixture was first concentrated using a rotavapor andwashed with 10% K₂CO₃ aqueous solution (100 mL). The mixture was thenextracted with DCM (2×100 mL) and the organic layers were combined,dried over Na₂SO₄ (20 g), filtered, and concentrated under reducedpressure to give a residue, which was then dissolved in anhydrous DCM(50 mL) and cooled down to 0° C. with an ice-water bath. Triethylamine(2.5 mL, 17.60 mmol) followed by bromoacetyl bromide (1.4 mL, 16.00mmol) were then added slowly at 0° C. After the addition was completed,the ice-water bath was removed and the mixture was stirred at ambienttemperature for 2 hours. Next, the reaction mixture was concentratedusing a rotavapor. The crude was dissolved in 10 mL DCM and purifiedwith a 220 g silica column using a gradient of 0-50% EtOAc/hexane for 40min under the flow rate at 60 mL/min. The product fractions werecollected and concentrated to yield Intermediate 5 (8.2 g, 79% yield) asa pale yellow solid. ¹H nmr (400 MHz, CDCl₃) δ: 4.23-4.24 (4H, m, OCH₂),3.93 (2H, s, ClCH₂), 3.60-3.69 (4H, m, NCH₂), 2.28-2.30 (4H, m, CH₂CO),1.57-1.59 (4H, m, CH₂CH₂CO), 1.24-1.27 (40H, m, CH₂), 0.85-0.89 (6H, m,CH₃).

Compound A4: Intermediate 5 (1.7 g, 2.57 mmol),(3S,4S)-dihydroxypyrrolidine (0.3 g, 2.57 mmol) in an oven-dried vial(40 mL) with a magnetic bar was added anhydrous DCM (20 mL). The mixturewas stirred at ambient temperature for 2 minutes to a clear solution.Triethylamine (0.4 mL, 2.57 mmol) was then added and the mixture wasstirred at ambient temperature overnight (17 hours). After removed thesolvent by rotavapor under high vacuum, the residue was added with 10%K₂CO₃ solution (50 mL) and extracted with DCM (2×50 mL). The organiclayers were then combined, dried over Na₂SO₄ (20 g), filtered, andconcentrated under reduced pressure. The crude was dissolved in 5 mL DCMand purified with a 24 g silica column using a gradient of 0-20%MeOH/DCM for 30 min under the flow rate at 20 mL/min. The productfractions were collected and concentrated to yield Compound A4 (870 mg,51% yield) as a white solid. ¹H nmr (400 MHz, CDCl₃) δ: 4.09-4.21 (6H,m, OCH₂, OCH), 3.56-3.63 (6H, m, NCH₂, COCH₂N), 3.28-3.29 (2H, m,NCH₂CH), 2.74-2.76 (2H, m, NCH₂CH), 2.26-2.30 (4H, m, CH₂CO), 1.59-1.60(4H, m, CH₂CH₂CO), 1.24-1.27 (40H, m, CH₂), 0.85-0.88 (6H, m, CH₃).

Example 4

A scheme for the preparation of Compound B8 is shown in FIG. 4.

Compound B8: Intermediate 4 (1.0 g, 1.60 mmol) in an oven-dried vial (20mL) with a magnetic bar was added anhydrous DCM (3 mL). The mixture wasstirred for 2 minutes to a clear solution. TFA (3 mL) was then added andthe mixture was stirred at ambient temperature for 2 hours. The reactionmixture was first concentrated using a rotavapor and washed with 10%K₂CO₃ aqueous solution (20 mL). The mixture was then extracted with DCM(2×20 mL) and the organic layers were combined, dried over Na₂SO₄ (2 g),filtered, and concentrated under reduced pressure to give anintermediate, which was then dissolved in anhydrous DCM (7 mL). Glutaricanhydride (0.3 g, 2.40 mmol) followed by triethylamine (0.3 mL, 2.40mmol) were then added at ambient temperature. The mixture was stirred atambient temperature overnight (17 hours). Next, the reaction mixture wasconcentrated using a rotavapor. The crude was dissolved in 2 mL DCM andpurified with a 24 g silica column using a gradient of 0-10% MeOH/DCMfor 20 min under the flow rate at 25 mL/min. The product fractions werecollected and concentrated to yield Compound B8 (667 mg, 65% yield) as awhite solid. ¹H nmr (400 MHz, CDCl₃) δ: 4.18-4.23 (4H, m, OCH₂),3.59-3.62 (4H, m, NCH₂), 2.44-2.49 (4H, m, CH₂CO), 2.29-2.31 (4H, m,CH₂CO), 1.97-2.00 (4H, m, CH₂CO), 1.57-1.62 (8H, m, CH₂CH₂CO, CH₂),1.26-1.31 (40H, m, CH₂), 0.87-0.89 (6H, m, CH₃).

Example 5

A scheme for the preparation of Compound A9 is shown in FIG. 5.

Compound A9: Intermediate 5 (2.4 g, 3.72 mmol), 3-azetidinemethanolhydrochloride (1.0 g, 8.09 mmol) in an oven-dried vial (40 mL) with amagnetic bar was added anhydrous DCM (20 mL) and DMSO (2 mL). Themixture was stirred at ambient temperature for 2 minutes to a clearsolution. Triethylamine (1.6 mL, 11.12 mmol) was then added and themixture was stirred at ambient temperature for 1 hours. The reaction wasquenched with saturated 10% K₂CO₃ solution (100 mL) and extracted withDCM (2×50 mL). The organic layers were then combined, dried over Na₂SO₄(20 g), filtered, and concentrated under reduced pressure. The crude wasdissolved in 5 mL DCM and purified with a 40 g silica column using agradient of 0-15% MeOH/DCM for 30 min under the flow rate at 20 mL/min.The product fractions were collected and concentrated to yield CompoundA9 (1.2 g, 49% yield) as a pale yellow solid. ¹H nmr (400 MHz, CDCl₃) δ:4.18-4.21 (4H, m, OCH₂), 3.76-3.77 (2H, m, CH₂OH), 3.60-3.50 (4H, m,NCH₂), 3.46-3.47 (2H, m, NCH₂), 3.38 (2H, s, COCH₂N), 3.25-3.26 (2H, m,NCH₂), 2.64-2.70 (1H, m, CH), 2.28-2.31 (4H, m, CH₂CO), 1.59 (4H, m,CH₂CH₂CO), 1.24-1.26 (40H, m, CH₂), 0.85-0.87 (6H, m, CH₃).

Example 6

A scheme for the preparation of Compound AA is shown in FIG. 6.

Compound AA: Intermediate 3 (460 mg, 0.61 mmol), 1-piperazinepropanol(88.5 m g, 0.61 mmol) in an oven-dried vial (40 mL) with a magnetic barwas added anhydrous DCM (10 mL). The mixture was stirred at ambienttemperature for 2 minutes to a clear solution. Triethylamine (0.1 mL,0.74 mmol) was then added and the mixture was stirred at ambienttemperature overnight (17 hours). The reaction was quenched with 10%K₂CO₃ solution (50 mL) and extracted with DCM (2×50 mL). The organiclayers were then combined, dried over MgSO₄ (5 g), filtered, andconcentrated under reduced pressure. The crude was dissolved in 5 mL DCMand purified with a 12 g silica column using a gradient of 0-20%MeOH/DCM for 30 min under the flow rate at 20 mL/min. The productfractions were collected and concentrated to yield Compound AA (389 mg,78% yield) as a colorless oil. ¹H nmr (400 MHz, CDCl₃) δ: 5.29-5.34 (8H,m, CH═), 4.20-4.23 (4H, m, OCH₂), 3.58-3.80 (6H, m, CH₂OH, NCH₂), 3.21(2H, s, COCH₂N), 2.75-2.78 (4H, m, ═CHCH₂CH═), 2.35-2.65 (8H, m, NCH₂),2.27-2.31 (4H, m, CH₂CO), 2.02-2.06 (8H, m, ═CHCH₂), 1.70-1.73 (4H, m,CH₂), 1.59-1.61 (4H, m, CH₂CH₂CO), 1.25-1.36 (28H, m, CH₂), 0.87-0.90(6H, m, CH₃).

Example 7

A scheme for the preparation of Compound A5 is shown in FIG. 7.

Compound A5: Intermediate 3 (200 mg, 0.27 mmol),trans-3,4-pyrrolidinediol (28 mg, 0.27 mmol) in an oven-dried vial (40mL) with a magnetic bar was added anhydrous DCM (10 mL) and DMSO (1 mL).The mixture was stirred at ambient temperature for 2 minutes to a clearsolution. Triethylamine (50 mL, 0.32 mmol) was then added and themixture was stirred at ambient temperature overnight (17 hours). Thereaction was quenched with 10% K₂CO₃ solution (50 mL) and extracted withDCM (2×50 mL). The organic layers were then combined, dried over MgSO₄(5 g), filtered, and concentrated under reduced pressure. The crude wasdissolved in 5 mL DCM and purified with a 12 g silica column using agradient of EtOAc for 5 min and 0-30% MeOH/DCM for 30 min under the flowrate at 20 mL/min. The product fractions were collected and concentratedto yield Compound A5 (389 mg, 78% yield) as a colorless liquid. ¹H nmr(400 MHz, CDCl₃) δ: 5.29-5.39 (8H, m, CH═), 4.20-4.25 (4H, m, OCH₂),4.10-4.11 (2H, m, CHOH), 3.55-3.62 (4H, m, NCH₂, COCH₂N), 3.28-3.31 (2H,m, NCH₂), 2.75-2.76 (6H, m, NCH₂, ═CHCH₂CH═), 2.60 (2H, m, NCH₂),2.27-2.32 (4H, m, CH₂CO), 2.02-2.06 (8H, m, ═CHCH₂), 1.60-1.61 (4H, m,CH₂CH₂CO), 1.25-1.36 (28H, m, CH₂), 0.87-0.90 (6H, m, CH₃).

Example 8

A scheme for the preparation of Compound A1 is shown in FIG. 8.

Compound A1: Intermediate 3 (1.0 g, 1.33 mmol), 3-azetidinemethanolhydrochloride (0.3 g, 2.66 mmol) in an oven-dried vial (40 mL) with amagnetic bar was added anhydrous DCM (20 mL) and DMSO (2 mL). Themixture was stirred at ambient temperature for 2 minutes to a clearsolution. Triethylamine (0.7 mL, 5.32 mmol) was then added and themixture was stirred at ambient temperature overnight (17 hours). Thereaction was quenched with 10% K₂CO₃ solution (50 mL) and extracted withDCM (2×50 mL). The organic layers were then combined, dried over Na₂SO₄(5 g), filtered, and concentrated under reduced pressure. The crude wasdissolved in 5 mL DCM and purified with a 24 g silica column using agradient of 0-50% EtOAc/hexane for 10 min and 0-15% MeOH/DCM for 20 minunder the flow rate at 20 mL/min. The product fractions were collectedand concentrated to yield Compound A1 (130 mg, 13% yield) as a paleyellow liquid. ¹H nmr (400 MHz, CDCl₃) δ: 5.29-5.35 (8H, m, CH═),4.18-4.21 (4H, m, OCH₂), 3.40-3.77 (12H, m, CH₂OH, CONCH₂, NCH₂,NCH₂CO), 2.74-2.77 (5H, m, CH, ═CHCH₂CH═), 2.28-2.30 (4H, m, CH₂CO),2.04-2.05 (8H, m, ═CHCH₂), 1.60 (4H, m, CH₂CH₂CO), 1.24-1.26 (40H, m,CH₂), 0.85-0.87 (6H, m, CH₃).

Example 9

A scheme for the preparation of Compound D22 is shown in FIG. 9.

Intermediate 6: Intermediate 1 (11.4 g, 15.6 mol) in an oven-dried flask(100 mL) with a magnetic bar was added anhydrous DCM (10 mL). Themixture was stirred for 2 minutes to a clear solution. TFA (10.0 mL) wasthen added and the mixture was stirred at ambient temperature for 2hours. During stirring, the solution color turned from clear to red.Next, the reaction mixture was first concentrated using a rotavapor andwashed with 10% K₂CO₃ aqueous solution (50 mL). The mixture was thenextracted with DCM (2×50 mL) and the organic layers were combined, driedover Na₂SO₄ (5 g), filtered, and concentrated under reduced pressure togive an oily residue, which was then dissolved in anhydrous DCM (100 mL)and added Fmoc-Glu(OtBu)-OH (6.6 g, 15.60 mmol), EDC (4.5 g, 23.40 mmol)and DMAP (0.4 g, 3.10 mmol) in sequence. The mixture was stirred atambient temperature overnight (17 hours). Next day, the reaction mixturewas washed with brine (100 mL), extracted with DCM (2×50 mL), dried overNa₂SO₄, filtered, and concentrated by rotavapor. The crude was dissolvedin 5 mL DCM and purified with a 220 g silica column using a gradient of0-50% EtOAc/hexane for 30 min under the flow rate at 50 mL/min. Theproduct fractions were collected and concentrated to yield Intermediate6 (8.3 g, 47% yield) as a clear liquid. ¹H nmr (400 MHz, CDCl₃) δ:7.75-7.76 (2H, d, J=6.4 Hz, ArH), 7.59 (2H, t, J=4.8 Hz, ArH), 7.38-7.41(2H, m, ArH), 7.26-7.33 (2H, m, ArH), 5.62-5.64 (1H, d, J=6.8 Hz,COCH₂), 5.30-5.39 (8H, m, OCH₂, ═CH), 4.76-4.79 (1H, m, COCH₂),4.11-4.41 (6H, m, CH, OCH₂), 3.35-3.98 (6H, m, NCH₂), 2.74-2.78 (4H, m,═CHCH₂CH═), 2.25-2.36 (8H, m, CH₂, CH₂CO), 2.00-2.06 (8H, m, ═CHCH₂),1.54-1.77 (4H, m, CH₂CH₂CO), 1.45 (9H, s, O(CH₃)₃), 1.24-1.37 (28H, m,CH₂), 0.87-0.90 (6H, m, CH₃).

Intermediate 7: Intermediate 6 (4.3 g, 4.14 mmol) was dissolved inacetonitrile (18 mL) in an oven-dried vial (100 mL) with a magnetic bar,followed by adding piperidine (2 mL). The mixture was stirred at ambienttemperature for 2 hours. Then solvent was removed under vacuum. Thecrude was dissolved in 5 mL DCM and purified with a 80 g silica columnusing a gradient of 0-5% MeOH/DCM for 30 min under the flow rate at 40mL/min. The product fractions were collected and concentrated to yieldIntermediate 7 (2.1 g, 64% yield) as a clear liquid. LC-MS analysis hasconfirmed the product—m/z of [M+H]⁺=816.17.

Intermediate 8: To Intermediate 7 (2.1 g, 2.58 mol) in an oven-driedflask (100 mL) with a magnetic bar were added anhydrous acetonitrile (30mL), sodium cyanoborohydride (0.9 g, 1.48 mmol), formaldehyde (37% inwater, 50 mL) and acetic acid (2 mL) in sequence. The mixture wasstirred for 2 hours. The reaction mixture was then treated with 10%K₂CO₃ aqueous solution (50 mL) and extracted with DCM (2×50 mL). Theorganic layers were combined, dried over Na₂SO₄ (5 g), filtered, andconcentrated under reduce pressure. The crude was dissolved in 2 mL DCMand purified with a 40 g silica column using a gradient of 0-10%MeOH/DCM for 30 min under the flow rate at 30 mL/min. The productfractions were collected and concentrated to yield Intermediate 8 (1.4g, 64% yield) as a clear liquid. LC-MS analysis has confirmed theproduct—m/z of [M+H]⁺=844.22.

Compound D22: Intermediate 8 (1.4 g, 1.66 mmol) in an oven-dried flask(100 mL) with a magnetic bar was added anhydrous DCM (10 mL). Themixture was stirred for 2 minutes to a clear solution. TFA (10.0 mL) wasthen added and the mixture was stirred at ambient temperature for 4hours. Then the reaction mixture was concentrated with a rotavapor. Thecrude was dissolved in 5 mL DCM and purified with a 24 g silica columnusing a gradient of 0-15% MeOH/DCM for 25 min under the flow rate at 25mL/min. The product fractions were collected and concentrated to yieldLp09 (200 mg, 15% yield) as a clear liquid. ¹H nmr (400 MHz, CDCl₃) δ:5.30-5.39 (8H, m, OCH₂, ═CH), 4.59-4.61 (1H, m, COCHN), 4.23-4.34 (4H,m, OCH₂), 3.54-3.77 (4H, m, NCH₂), 2.90 (6H, s, NCH₃), 2.76 (4H, t,J=5.2 Hz, ═CHCH₂CH═), 2.50-2.55 (2H, m, CH₂CO), 2.31-2.34 (4H, m,CH₂CO), 2.22-2.27 (2H, m, CH₂), 2.03-2.07 (8H, m, ═CHCH₂), 1.59-1.60(4H, m, CH₂CH₂CO), 1.26-1.37 (28H, m, CH₂), 0.88-0.90 (6H, m, CH₃).

Example 10

A scheme for the preparation of Compounds A7 and A8 is Shown in FIG. 10.

Intermediate 9: Intermediate 1 (1.0 g, 1.37 mmol) in an oven-dried flask(100 mL) with a magnetic bar was added anhydrous DCM (10 mL). Themixture was stirred for 2 minutes to a clear solution. TFA (10.0 mL) wasthen added and the mixture was stirred at ambient temperature for 2hours. During stirring, the solution color turned from clear to red.Next, the reaction mixture was first concentrated using a rotavapor andwashed with 10% K₂CO₃ aqueous solution (50 mL). The mixture was thenextracted with DCM (2×50 mL) and the organic layers were combined, driedover Na₂SO₄ (5 g), filtered, and concentrated under reduced pressure togive Intermediate 9 as an oil, which was then used without furtherpurification.

Intermediate 10: To 2-(dimethylamino)ethan-1-ol (0.1 mL, 1.39 mmol) inanhydrous DCM (10 mL) was added 4-nitrophenyl carbonochloridate (0.3 g,1.39 mmol) followed by DIEA (0.5 mL, 2.76 mmol). The mixture was stirredat ambient temperature overnight (17 hours) to give the Intermediate 10solution, which was used directly without further purification.

Compound A7: Intermediate 9 was dissolved in DCM (10 mL) and transferredinto solution of Intermediate 10 in a flask. The mixture was stirred atambient temperature overnight (17 hours). Next day, the reaction mixturewas concentrated using a rotavapor. The crude was dissolved in 2 mL ofDCM and purified with a 24 g silica column using a gradient of 0-50%EtOAc/hexane for 10 min, 2-20% MeOH/DCM for 20 min under the flow rateat 25 mL/min. The product fractions were collected and concentrated toyield Compound A7 (540 mg, 54% yield) as a clear yellow liquid. ¹H nmr(400 MHz, CDCl₃) δ: 5.30-5.40 (8H, m, CH═), 4.20-4.40 (6H, m, OCH₂),3.45-3.55 (6H, m, NCH₂), 2.75-2.78 (4H, m, ═CHCH₂CH═), 2.25-2.30 (10H,m, NCH₃, CH₂CO), 2.03-2.05 (8H, m, ═CHCH₂), 1.56-1.60 (4H, m, CH₂CH₂CO),1.29-1.33 (28H, m, CH₂), 0.86-0.90 (6H, m, CH₃).

Compound A8: Compound A7 (160 mg, 0.22 mmol) was added to methyl iodide(2 mL) in an oven-dried vial (20 mL) with a magnetic bar. The mixturewas stirred at ambient temperature for 2 hours. Next, methyl iodide wasremoved under vacuum. The crude was dissolved in 1 mL DCM and purifiedwith a 12 g silica column using a gradient of 2-20% MeOH/DCM for 30 minunder the flow rate at 20 mL/min. The product fractions were collectedand performed iodide to chloride anion exchange using resin Amberst 26.The resulting solution was finally concentrated under reduced pressureto give Compound A8 (100 mg, 59% yield) as a clear liquid. ¹H nmr (400MHz, CDCl₃) δ: 5.29-5.34 (8H, m, CH═), 4.60-4.65 (2H, m, OCH₂),4.10-4.20 (6H, m, NCH₂, OCH₂), 3.50-3.55 (13H, m, NCH₃, NCH₂), 2.75-2.78(4H, m, ═CHCH₂CH═), 2.25-2.30 (4H, m, CH₂CO), 2.02-2.06 (8H, m, ═CHCH₂),1.59-1.61 (4H, m, CH₂CH₂CO), 1.25-1.36 (28H, m, CH₂), 0.87-0.90 (6H, m,CH₃).

Example 11

A scheme for the preparation of Compounds C3 and C2 is Shown in FIG. 11.

Intermediate 11: Boc-beta-H-Asp(OBz)-OH (35.0 g, 10.20 mmol) wasdissolved in MeOH (45 mL) in a round-bottomed flask flushed with Argongas. 10% Pd/C (350 mg) was added and the flask was flushed with Argongas once again. Next, all air was removed via vacuum pump and a balloonfilled with hydrogen gas was attached. Reaction mixture was allowed tostir for ˜2 hours at ambient temperature. The hydrogen balloon wasremoved and the mixture was then filtered out Pd/C catalyst through apad of celite, followed by rinsing with copious amounts of MeOH. Thefiltrate was finally concentrated by a rotovap to yield Intermediate 11(2.5 g, 100% yield), which was used without further purification.

Intermediate 12: Intermediate 11 (2.5 g, 10.11 mmol), EDC (5.8 g, 30.30mmol), and DMAP (494 mg, 4.04 mmol) in an oven-dried flask (200 mL) witha magnetic bar was added anhydrous DCM (50 mL). The mixture was stirredat ambient temperature for ˜5 minutes to a clear solution. Linoleylalcohol (6.5 g, 24.20 mmol) was then added and the mixture was stirredat room temperature overnight. The reaction was finally quenched withH₂O (50 mL) and extracted with DCM twice (2×50 mL). Organic layers werecombined, dried over MgSO₄, and filtered. The filtrate was concentratedunder reduced pressure. The crude purified by flash chromatographypurification system (80 g silica gel column) using a gradient of hexanefor 2 min, then 0-25% EtOAc/hexane for 15 min, then 25% EtOAc/hexane for5 min, then 75% EtOAc/hexane for 5 min under the flow rate at 60 mL/min.The product fractions were collected and concentrated to yieldIntermediate 12 (7.0 g, 93% yield) as a clear liquid. ¹H nmr (400 MHz,CDCl₃) δ: 5.38-5.31 (8H, m, CH═), 4.28 (1H, bs, NH), 4.08-4.05 (4H, m,OCH₂), 2.78-2.75 (4H, m, ═CHCH₂CH═), 2.69-2.59 (4H, m, COCH₂N),2.06-2.02 (4H, m, CH₂CH₂CH), 1.62-1.58 (4H, m, CH₂CH₂O), 1.43 (9H, s,C(CH₃)₃), 1.35-1.26 (36H, m, CH₂), 0.90-0.86 (6H, m, CH₃).

Intermediate 13: Intermediate 12 (2.3 g, 3.09 mmol) was dissolved in DCM(20 mL) and cooled in an ice-bath. TFA (20 mL) was added and the mixturewas allowed to stir for ˜1 hour under a blanket of argon gas.Afterwards, material was concentrated in vacuo. The residue wasdissolved in DCM (20 mL) and then 10% K₂CO₃ (20 mL) was added. Afterstirred the mixture in an ice bath for ˜½ hour, it was partitioned,checking the pH of the aqueous to ensure it was basic. The turbidaqueous layer was extracted with DCM (3×20 mL). The combined organiclayers was added MgSO₄, stirred once again in the ice bath for ˜20minutes, and filtered. The filtrate, Intermediate 13, was then carriedforward without any further refinement (2.0 g, assumed quantitativeyield).

Compound C3: N,N-Dimethyl glycine HCl salt (260 mg, 1.86 mmol), EDC (447mg, 2.33 mmol), and DMAP (38 mg, 0.31 mmol) in an oven-dried flask (50mL) with a magnetic bar was added anhydrous DCM (15 mL). The mixture wasstirred at ambient temperature for ˜5 minutes. Intermediate 13 (1.0 g,1.55 mmol) was then added and the mixture was stirred at roomtemperature overnight. The reaction was finally quenched with H₂O (50mL) and extracted with DCM twice (2×50 mL). Organic layers werecombined, dried over MgSO₄, and filtered. The filtrate was concentratedunder reduced pressure. The crude purified by flash chromatographypurification system (40 g silica gel column) using a gradient of hexanefor 1 min, then 0-50% EtOAc/hexane for 25 min, then 50% EtOAc/hexane for5 min under the flow rate at 40 mL/min. The product fractions werecollected and concentrated to yield Lp12 (890 mg, 79% yield) as a clearliquid. ¹H nmr (400 MHz, CDCl₃) δ: 7.76-7.74 (1H, d, NH), 5.40-5.30 (8H,m, CH═), 4.28 (1H, m, CHN), 4.08-4.05 (4H, m, OCH₂), 2.92 (2H, s,COCH₂N), 2.78-2.75 (4H, m, ═CHCH₂CH═), 2.732.62 (4H, m, COCH₂N), 2.27(6H, s, N(CH₃)₂), 2.06-2.02 (4H, m, CH₂CH₂CH), 1.62-1.58 (4H, m,CH₂CH₂O), 1.43 (9H, s, C(CH₃)₃), 1.35-1.26 (36H, m, CH₂), 0.90-0.86 (6H,m, CH₃).

Compound C2: Compound C3 (890 mg, 1.22 mmol) was dissolved inacetonitrile (9 mL) and iodomethane (1 mL) was added. Vial was flushedwith argon gas and allowed to stir at 50° C. overnight. Next day, thereaction mixture was concentrated in vacuo and purified by flashchromatography purification system (40 g silica gel column) using agradient of DCM for 2 min, then 0-8% MeOH/DCM for 20 min, then 8%MeOH/DCM for 5 min under the flow rate at 40 mL/min. The productfractions were collected and concentrated and subjected to Amberlyst A26Anion Exchange resin to yield Compound C2 (623 mg, 66% yield) as oil. ¹Hnmr (400 MHz, CDCl₃) δ: 9.69-9.66 (1H, d, NH), 5.40-5.30 (8H, m, CH═),4.67 (1H, m, CHN), 4.57 (2H, s, COCH₂N), 4.08-4.05 (4H, m, OCH₂), 3.43(9H, s, N(CH₃)₃), 2.78-2.75 (4H, m, ═CHCH₂CH═), 2.73-2.62 (4H, m,COCH₂N), 2.06-2.02 (4H, m, CH₂CH₂CH), 1.62-1.58 (4H, m, CH₂CH₂O), 1.43(9H, s, C(CH₃)₃), 1.35-1.26 (36H, m, CH₂), 0.90-0.86 (6H, m, CH₃).

Example 12

A scheme for the preparation of Compound DD is shown in FIG. 12.

Intermediate 14: Intermediate 12 (4.4 g, 5.93 mmol) was dissolved in DCM(20 mL) and cooled in an ice-bath. TFA (20 mL) was added and the mixturewas allowed to stir for ˜1 hour under a blanket of Argon gas. Thereaction mixture was then concentrated in vacuo. The residue wasdissolved in DCM (20 mL) and then 10% K₂CO₃ (20 mL) was added. Afterstirred the mixture in an ice bath for ˜½ hour, it was partitioned,checking the pH of the aqueous to ensure it was basic. The turbidaqueous layer was extracted with DCM (3×20 mL). The combined organiclayers was added MgSO₄, stirred once again in the ice bath for ˜20minutes, and filtered. The filtrate was then added diphosgene (1.1 mL,8.90 mmol). The reaction mixture was allowed to stir overnight atambient temperatures under a blanket of argon gas. Next day, DCM andexcess diphosgene were removed in vacuo. The residue, Intermediate 14,was dried fully before carrying forward without any further refinement(4.6 g, assumed quantitative yield).

Compound DD: 2-(Dimethylamino) ethanethiol HCl salt (2.1 g, 14.90 mmol)was suspended in DCM (25 mL) and added to Intermediate 14 (2.3 g, 2.97mmol) in DCM (25 mL). TEA (2.7 mL, 19.30 mmol) was added slowly to themixture. Reaction was allowed to stir overnight at ambient temperatureunder a blanket of argon gas. Next day, the reaction mixture was dilutedwith DCM (100 mL) and washed with H₂O (100 mL) followed by 10% K₂CO₃(100 mL). Back-extraction was performed for both aqueous washes with DCM(2×40 mL). The organic layers were combined, dried with MgSO₄, filtered,and concentrated in vacuo. The crude was purified by flashchromatography purification system (80 g silica gel column) using agradient of 10% EtOAc/hexane for 3 min, then 10-100% EtOAc/hexane for 15min, then EtOAc for 5 minutes under the flow rate at 60 mL/min. Theproduct fractions were collected and concentrated to yield Compound DD(300 mg, 39% yield) as a clear liquid. ¹H nmr (400 MHz, CDCl₃) δ:6.53-6.51 (1H, d, NH), 5.34-5.30 (8H, m, CH═), 4.57 (1H, m, CHN),4.08-4.05 (4H, m, OCH₂), 2.75-2.68 (2H, m, SCH₂CH₂), 2.78-2.75 (4H, m,═CH—CH₂CH═), 2.73-2.62 (4H, m, COCH₂N), 2.30-2.25 (2H, m, SCH₂CH₂) 2.15(6H, s, N(CH₃)₂), 2.06-2.02 (4H, m, CH₂CH₂CH), 1.62-1.58 (4H, m,CH₂CH₂O), 1.43 (9H, s, C(CH₃)₃), 1.35-1.26 (36H, m, CH₂), 0.90-0.86 (6H,m, CH₃).

Example 13

A scheme for the preparation of Compound E4 is shown in FIG. 13.

Compound E4: Intermediate 14 (6.4 g, 8.22 mmol) was suspended in DCM (75mL) in a round bottom flask (500 mL) with a magnetic stir bar.2-(Dimethylamino) ethanol (4.1 mL, 41.1 mmol) followed by TEA (7.4 mL,53.4 mmol) was added slowly to the mixture. Reaction was allowed to stirovernight at ambient temperature under a blanket of argon gas. Next day,the reaction mixture was diluted with DCM (100 mL) and washed with H₂O(100 mL) followed by 10% K₂CO₃ (100 mL). Back-extraction was performedfor both aqueous washes with DCM (2×40 mL). The organic layers werecombined, dried with MgSO₄, filtered, and concentrated in vacuo. Thecrude was purified by flash chromatography purification system (80 gsilica gel column) using a gradient of 10% EtOAc/hexane for 3 min, then10-100% EtOAc/hexane for 15 min, then EtOAc for 5 minutes under the flowrate at 60 mL/min. The product fractions were collected and concentratedto yield Compound E4 (3.3 g, 53% yield) as a clear liquid. ¹H nmr (400MHz, CDCl₃) δ: 6.53-6.51 (1H, bs, NH), 5.40-5.20 (8H, m, CH═), 4.35 (1H,m, CHN), 4.20-4.10 (2H, m, COOCH₂), 4.10-4.00 (4H, m, COOCH₂), 2.78-2.75(4H, m, ═CHCH₂CH═), 2.732.62 (4H, m, COCH₂N), 2.60-2.50 (2H, m, OCH₂CH₂)2.25 (6H, s, N(CH₃)₂), 2.06-2.02 (4H, m, CH₂CH₂CH), 1.62-1.58 (4H, m,CH₂CH₂O), 1.43 (9H, s, C(CH₃)₃), 1.35-1.26 (36H, m, CH₂), 0.90-0.86 (6H,m, CH₃).

Example 14

A scheme for the preparation of Compound CA is shown in FIG. 14.

Intermediate 15: Intermediate 13 (1.0 g, 1.55 mmol) was dissolved inanhydrous DCM (15 mL) and bromoacetyl bromide (135 μL, 1.55 mmol)followed by TEA (238 μL, 1.71 mmol) were then added slowly. After theaddition was completed, the mixture was stirred at ambient temperatureovernight. Next day, the mixture was diluted with DCM (50 mL) and washedwith H₂O (50 mL) and 10% K₂CO₃ (50 mL). Back-extraction was performedfor both aqueous washes with DCM (2×25 mL). The organic layers werecombined, dried with MgSO₄, filtered, and concentrated in vacuo. Thecrude was purified with a 40 g silica column on flash chromatographysystem equipped with ESLD detector using a gradient of hexane for 0.5min, then 0-50% EtOAc/hexane 30 min gradient followed by 50%EtOAc/hexane for 5 min under the flow rate at 40 mL/min. The productfractions were collected and concentrated to yield Intermediate 15 (950mg, 80% yield) as a colorless liquid. ¹H nmr (400 MHz, CDCl₃) δ:7.44-7.42 (1H, bs, NH), 5.40-5.32 (8H, m, CH═), 4.62-4.57 (1H, m, CHN),4.10-4.07 (4H, m, COOCH₂), 3.83 (2H, s, CH₂Br), 2.78-2.75 (4H, m,═CH—CH₂CH═), 2.73-2.62 (4H, m, COCH₂N), 2.60-2.50 (2H, m, OCH₂CH₂) 2.25(6H, s, N(CH₃)₂), 2.06-2.02 (4H, m, CH₂CH₂CH), 1.62-1.58 (4H, m,CH₂CH₂O), 1.43 (9H, s, C(CH₃)₃), 1.35-1.26 (36H, m, CH₂), 0.90-0.86 (6H,m, CH₃).

Compound CA: Intermediate 16 (400 mg, 0.52 mmol) was suspended in DCM(10 mL) in a round bottom flask (50 mL) with a magnetic stir bar and1-(2-hydroxyethyl)piperazine (70 μL, 0.58 mmol) followed by TEA (91 μL,0.65 mmol) was added. Mixture was stirred overnight at ambienttemperature under a blanket of argon gas. Next day, the mixture wasdiluted with DCM (25 mL) and washed with H₂O (25 mL) followed by 10%K₂CO₃ (25 mL). Back-extraction was performed for both aqueous washeswith DCM (2×25 mL). The organic layers were combined, dried with MgSO₄,filtered, and concentrated in vacuo. The crude purified by flashchromatography purification system (40 g silica gel column) using a DCMfor 1 min, then 10% MeOH/DCM for 15 min under the flow rate at 40mL/min. The product fractions were collected and concentrated to yieldCompound CA (400 mg, 94% yield) as a clear liquid. ¹H nmr (400 MHz,CDCl₃) δ: 7.82-7.80 (1H, bs, NH), 5.40-5.29 (8H, m, CH═), 4.61-4.57 (1H,m, CHN), 4.07-4.04 (4H, m, COOCH₂), 3.63-3.61 (2H, m, CH₂CH₂OH), 2.97(2H, s, COCH₂N), 2.78-2.75 (4H, m, ═CHCH₂CH═), 2.73-2.62 (4H, m,COCH₂N), 2.62-2.59 (2H, m, NCH₂CH₂OH), 2.59-2.50 (8H, m, N(CH₃)₂N),2.06-2.02 (8H, m, CH₂CH₂CH), 1.62-1.58 (4H, m, CH₂CH₂O), 1.35-1.26 (32H,m, CH₂), 0.90-0.86 (6H, m, CH₃).

Example 15

A scheme for the preparation of Compound D1 is shown in FIG. 15.

Compound D1: Intermediate 15 (500 mg, 0.653 mmol) was suspended in DCM(8.5 mL) in a scintillation vial with a magnetic stir bar.Cis-pyrrolidine-3,4-diol HCl salt (100 mg, 0.72 mmol) in DMSO (1 mL) wasadded followed by TEA (229 μL, 1.64 mmol). The mixture was stirredovernight at ambient temperature under a blanket of argon gas. Next day,the mixture was diluted with DCM (25 mL) and washed with H₂O (25 mL)followed by 10% K₂CO₃ (25 mL). Back-extraction was performed for bothaqueous washes with DCM (2×10 mL). The organic layers were combined,dried with MgSO₄, filtered, and concentrated in vacuo. The crude waspurified by flash chromatography purification system (40 g silica gelcolumn) using a DCM for 1 min, then 10% MeOH/DCM for 15 min under theflow rate at 40 mL/min. The product fractions were collected andconcentrated to yield Compound D1 (437 mg, 87% yield) as a clear liquid.¹H nmr (400 MHz, CDCl₃) δ: 7.82-7.80 (1H, bs, NH), 5.40-5.29 (8H, m,CH═), 4.61-4.57 (1H, m, CHN), 4.07-4.04 (4H, m, COOCH₂), 3.63-3.61 (2H,m, CH₂CH₂OH), 3.10 (2H, s, CH₂CHOH), 2.78-2.75 (4H, m, ═CHCH₂CH═),2.73-2.62 (4H, m, COCH₂N), 2.62-2.58 (4H, m, N(CH₂)₂CHOH), 2.06-2.02(8H, m, CH₂CH₂CH), 1.62-1.58 (4H, m, CH₂CH₂O), 1.35-1.29 (32H, m, CH₂),0.90-0.86 (6H, m, CH₃).

Example 16

A scheme for the preparation of Compound D7 is shown in FIG. 16.

Compound D7: Intermediate 15 (750 mg, 0.98 mmol) was suspended in DCM(10 mL) in a round bottom flask (50 mL) with a magnetic stir bar andAzetidine-3-yl-methanol HCl salt (149 mg, 1.18 mmol) was added followedby TEA (341 μL, 2.45 mmol). The mixture was stirred overnight at ambienttemperature under a blanket of argon gas. Next day, the mixture wasdiluted with DCM (25 mL) and washed with H₂O (25 mL) followed by 10%K₂CO₃ (25 mL). Back-extraction was performed for both aqueous washeswith DCM (2×10 mL). The organic layers were combined, dried with MgSO₄,filtered, and concentrated in vacuo. The crude purified by flashchromatography purification system (40 g silica gel column) using a DCMfor 2 min, then 10% MeOH/DCM for 20 min under the flow rate at 40mL/min. The product fractions were collected and concentrated to yieldCompound D7 (350 mg, 46% yield) as a clear liquid. ¹H nmr (400 MHz,CDCl₃) δ: 7.71-7.68 (1H, bs, NH), 5.35-5.33 (8H, m, CH═), 4.57-4.55 (1H,m, CHN), 4.07-4.04 (4H, m, COOCH₂), 3.77 (2H, s, COCH₂N), 3.38-3.35 (2H,m, CHCH₂OH), 3.20-3.00 (4H, m, N(CH₃)₂CH), 2.78-2.75 (4H, m, ═CHCH₂CH═),2.73-2.62 (4H, m, COCH₂N), 2.06-2.02 (8H, m, CH₂CH₂CH), 1.62-1.58 (4H,m, CH₂CH₂O), 1.35-1.26 (32H, m, CH₂), 0.90-0.86 (6H, m, CH₃).

Example 17

A scheme for the preparation of Compound F6 is shown in FIG. 17.

Intermediate 16: N,N-Dimethyl-1,4-butane-diamine (1.0 g, 8.60 mmol) wasdissolved in MeOH (15 mL). Mixture was placed in an ice-bath and(tert-butyl)-dimetholsiloxy)-acetaldehyde (1.7 mL, 9.03 mmol) was addedand stirred for 1 hour. NaBH₄ (522 mg, 13.8 mmol) was then added and themixture was stirred for another hour 0° C. Next, the reaction wasquenched with H₂O (1 mL) and concentrated in vacuo to yield Intermediate16. The material was carried forward with any further refinement (2.4 g,assumed quantitative yield).

Intermediate 17: Linoleic acid (1.2 g, 4.37 mmol), EDC (1.1 g, 5.46mmol), and DMAP (89 mg, 0.73 mmol) in an oven-dried flask (50 mL) with amagnetic bar was added anhydrous DCM (15 mL). The mixture was stirred atambient temperature for ˜5 minutes to a clear solution. Intermediate 16(1.0 g, 3.64 mmol) was then added and the mixture was stirred at roomtemperature overnight. The reaction was finally quenched with H₂O (50mL) and extracted with DCM twice (2×50 mL). Organic layers werecombined, dried over MgSO₄, and filtered. The filtrate was concentratedunder reduced pressure. The crude was purified by flash chromatographypurification system (24 g silica gel column) using a gradient of DCM for1 min, then 0-10% MeOH/DCM for 2 min, then 10% MeOH/DCM for 7 min, then10-30% MeOH/DCM for 2 min, then 30% MeOH/DCM for 7 min under the flowrate at 35 mL/min. The product fractions were collected and concentratedto yield Intermediate 17 (460 mg, 24% yield) as a clear liquid. Theproduct was verified with LC-MS prior to executing the next step.

Intermediate 18: 1.0M TBAF/THF (2.6 mL, 2.57 mmol) was added toIntermediate 17 (460 mg, 0.86 mmol) dissolved in THF (5 mL) in anoven-dried flask (50 mL) with a magnetic bar and the mixture was allowedto stir at ambient temperature overnight under a blanket of argon gas.Next day, the mixture was concentrated in vacuo and carried forwardwithout any further purification (361 mg, assumed quantitative yield).The product was verified with LC-MS prior to executing the next step.

Compound F6: Linoleic acid (289 mg, 1.0 mmol), EDC (247 mg, 1.29 mmol),and DMAP (21 mg, 0.171 mmol) in an oven-dried flask (50 mL) with amagnetic bar was added anhydrous DCM (20 mL). The mixture was stirred atambient temperature for ˜5 minutes to a clear solution. Intermediate 18(361 mg, 0.86 mmol) was then added and the mixture was stirred at roomtemperature overnight. Next day, the mixture was diluted with DCM (50mL) and washed with H₂O (50 mL) followed by 10% K₂CO₃ (50 mL).Back-extraction was performed for both aqueous washes with DCM (2×20mL). The organic layers were combined, dried with MgSO₄, filtered, andconcentrated in vacuo. The crude was purified by flash chromatographypurification system (80 g silica gel column) using a gradient of hexanefor 2 min, then 0-25% EtOAc/hexane for 15 min, then 25% EtOAc/hexane for5 min, then 75% EtOAc/hexane for 5 min under the flow rate at 60 mL/min.The product fractions were collected and concentrated to yield CompoundF6 (385 mg, 65% yield) as a clear liquid. ¹H nmr (400 MHz, CDCl₃) δ:5.40-5.30 (8H, m, CH═), 4.20-4.17 (2H, m, NCH₂CH₂O), 3.56-3.50 (2H, m,NCH₂CH₂O), 3.38-3.27 (2H, m, CO NCH₂), 2.77-2.75 (2H, m, ═CHCH₂CH═),2.66-2.56 (4H, m, N(CH₃)₂CH₂), 2.40-2.27 (10H, m, COCH₂, & N(CH₃)₂),2.06-2.02 (8H, m, CH₂CH₂CH), 1.85-1.58 (8H, m, CH₂CH₂CO &NCH₂(CH₂)₂CH₂N), 1.36-1.26 (28H, m, CH₂), 0.90-0.86 (6H, m, CH₃).

Example 18

A scheme for the preparation of Compounds F5 and F7 is Shown in FIG. 18.

Intermediate 19: 3-(Dimethylamino)-1-propylamine (4.4 mL, 30.50 mmol)was dissolved in MeOH (20 mL). Mixture was placed in an ice-bath and(tert-butyl-dimethylsilyl)acetaldehyde (6.1 mL, 32.00 mmol) was addedallowed to stir for 1 hour. NaBH₄ (1.9 g, 48.79 mmol) was then added andthe mixture was stirred for another hour at 0° C. Next, the reaction wasquenched with H₂O (5 mL), filtered out precipitate, and concentrated invacuo to yield Intermediate 19. Material was carried forward with anyfurther refinement (8.1 g, assumed quantitative yield).

Intermediate 20: Linoleic Acid (10.4 g, 37.10 mmol), EDC (8.9 g, 46.42mmol), and DMAP (755 mg, 6.22 mmol) in an oven-dried flask (200 mL) witha magnetic bar was added anhydrous DCM (50 mL). The mixture was stirredat ambient temperature for ˜5 minutes to a clear solution. Intermediate19 (8.1 g, 30.91 mmol) was then added and the mixture was stirred atroom temperature overnight. Next day, diluted with DCM (50 mL) andwashed with H₂O (50 mL) followed by 10% K₂CO₃ (50 mL). Back-extractionwas performed for both aqueous washes with DCM (2×25 mL). The organiclayers were combined, dried with MgSO₄, filtered, and concentrated invacuo. The crude purified by flash chromatography purification system(330 g silica gel column) using a gradient of DCM for 5 min, then 0-10%MeOH/DCM for 25 min, then 10% MeOH/DCM for 15 min under the flow rate at200 mL/min. The product fractions were collected and concentrated toyield Intermediate 20 (9.8 g, 60% yield) as a clear liquid. ¹H nmr (400MHz, CDCl₃) δ: 5.37-5.29 (4H, m, CH═), 3.75-3.67 (2H, m, NCH₂CH₂O),3.46-3.35 (4H, m, CONCH₂), 2.77-2.74 (2H, m, ═CHCH₂CH═), 2.36-2.23 (4H,m, COCH₂ & (CH₃)₂NCH₂), 2.21-2.20 (6H, d, (CH₃)₂N), 2.04-2.00 (4H, m,CH₂CH₂CH), 1.71-1.59 (2H, m, NCH₂CH₂CH₂N), 1.36-1.26 (14H, m, CH₂),0.89-0.86 (12H, s, SiC(CH₃)₃& CH₃), 0.36-0.28 (6H, m, Si(CH₃)₂).

Intermediate 21: 1.0 M TBAF/THF (55.2 mL, 55.21 mmol) was added toIntermediate 20 (9.6 g, 18.43 mmol) in an oven-dried flask (50 mL) witha magnetic bar and allowed to stir for three hours under a blanket ofargon gas. Then, the mixture was concentrated in vacuo and carriedforward with any further refinement (7.5 g, assumed quantitative yield).The product was verified with LC-MS prior to executing the next step.

Compound F5: Linoleic Acid (6.2 g, 22.13 mmol), EDC (5.3 g, 27.64 mmol),and DMAP (450 mg, 3.68 mmol) in an oven-dried flask (50 mL) with amagnetic bar was added anhydrous DCM (20 mL). The mixture was stirred atambient temperature for ˜5 minutes to a clear solution. Intermediate 21(7.5 g, 18.42 mmol) was then added and the mixture was stirred at roomtemperature overnight. Next day, diluted with DCM (50 mL) and washedwith H₂O (50 mL) flowed by 10% K₂CO₃ (50 mL). Back-extraction wasperformed for both aqueous washes with DCM (2×40 mL). The organic layerswere combined, dried with MgSO₄, filtered, and concentrated in vacuo.The crude purified by flash chromatography purification system (330 gsilica gel column) using a gradient of DCM for 10 min, then 0-5%MeOH/DCM for 5 min, then 5% MeOH/DCM for 15 min, then 5-20% MeOH/DCM for10 min, then 20% MeOH/DCM for 2 minutes under the flow rate at 200mL/min. The product fractions were collected and concentrated to yieldCompound F5 (8.0 g, 65% yield) as a clear liquid. ¹H nmr (400 MHz,CDCl₃) δ: 5.40-5.20 (8H, m, CH═), 4.20-4.17 (2H, m, NCH₂CH₂O), 3.90-4.10(2H, m, NCH₂CH₂O), 3.50-3.60 (2H, m, CONCH₂), 2.77-2.75 (4H, m,═CHCH₂CH═), 2.40-2.10 (14H, m, CH₂CH₂CH & N(CH₃)₂), 2.10-1.90 (8H, m,COCH₂, & N(CH₂)& OCOCH₂CH₂), 2.06-2.02 (4H, m, CH₂CH₂CH), 1.80-1.50 (4H,m, NCH₂CH₂CH₂N & NCOCH₂CH₂), 1.40-1.00 (28H, m, CH₂), 0.99-0.71 (6H, m,CH₃).

Compound F7: Compound F5 (6.1 g, 9.04 mmol) was dissolved inacetonitrile (18 mL) and methyl iodide (2 mL) was added. Vial wasflushed with argon gas and the mixture was allowed to stir at 40° C. for4 hours. Afterwards, the mixture was concentrated in vacuo and purifiedby flash chromatography with loading the crude oil directly onto 120 gsilica gel column and using a gradient of DCM for 2 min, then 0-10%MeOH/DCM for 10 min, then 10% MeOH/DCM for 5 min, then 10-15% MeOH/DCMfor 5 min, then 15% MeOH/DCM for 5 min under the flow rate at 85 mL/min.The product fractions were collected, concentrated, and subjected toAmberlyst A26 Anion Exchange resin to yield Compound F7 (3.2 g, 50%yield) as oil. ¹H nmr (400 MHz, CDCl₃) δ: 5.40-5.20 (8H, m, CH═),4.30-4.20 (2H, m, NCH₂CH₂O), 3.80-3.60 (4H, m, NCH₂CH₂O & CONCH₂),3.68-3.39 (9H, m, N(CH₃)₃), 2.77-2.73 (4H, m, ═CHCH₂CH═), 2.40-2.30 (2H,m, COCH₂), 2.30-2.20 (2H, m, (CH₃)₂NCH₂), 2.20-1.10 (2H, m, OCOCH₂CH₂),2.10-1.90 (8H, m, CH₂CH₂CH), 1.70-1.50 (4H, m, NCH₂CH₂CH₂N & NCOCH₂CH₂),1.40-1.10 (28H, m, CH₂), 0.90-0.85 (6H, m, CH₃).

Example 19

A scheme for the preparation of Compounds F8 and F9 is Shown in FIG. 19.

Intermediate 22: Myristic Acid (2.0 g, 8.82 mmol), EDC (2.1 g, 11.02mmol), and DMAP (180 mg, 1.47 mmol) in an oven-dried flask (100 mL) witha magnetic bar was added anhydrous DCM (20 mL). The mixture was stirredat ambient temperature for ˜5 minutes to a clear solution. Intermediate19 (1.9 g, 7.35 mmol) was then added and the mixture was stirred at roomtemperature overnight. Next day, the reaction mixture was diluted withDCM (50 mL) and washed with H₂O (50 mL) flowed by 10% K₂CO₃ (50 mL).Back-extraction was performed for both aqueous washes with DCM (2×25mL). The organic layers were combined, dried with MgSO₄, filtered, andconcentrated in vacuo. The crude was purified by flash chromatographypurification system (80 g silica gel column) using a gradient of DCM for1 min, then 0-10% MeOH/DCM for 15 min, then 10% MeOH/DCM for 5 min, then30% MeOH/DCM for 5 min under the flow rate at 60 mL/min. The productfractions were collected and concentrated to yield Intermediate 22 (1.5g, 39% yield) as a clear liquid. ¹H nmr (400 MHz, CDCl₃) δ: 3.75-3.67(2H, m, NCH₂CH₂O), 3.44-3.35 (4H, m, CONCH₂), 2.36-2.21 (4H, m, COCH₂ &(CH₃)₂NCH₂), 2.26-2.21 (6H, d, (CH₃)₂N), 1.71-1.59 (2H, m, NCH₂CH₂CH₂N),1.63-1.59 (2H, m, COCH₂CH₂), 1.28-1.24 (20H, m, CH₂), 0.88-0.85 (12H, m,SiC(CH₃)₃& CH₃), 0.38-0.30 (6H, m, Si(CH₃)₂).

Intermediate 23: 1.0 M TBAF/THF (6.4 mL, 6.36 mmol) was added toIntermediate 22 (1.0 g, 2.12 mmol) dissolved in THF (5 mL) in anoven-dried flask (25 mL) with a magnetic bar and allowed to stir forthree hours under a blanket of argon gas. Then, the mixture wasconcentrated in vacuo and carried forward with any further refinement(728 mg, assumed quantitative yield). The product was verified withLC-MS prior to executing the next step.

Compound F8: Linoleic Acid (659 mg, 2.35 mmol), EDC (631 mg, 3.29 mmol),and DMAP (54 mg, 0.44 mmol) in an oven-dried flask (25 mL) with amagnetic bar was added anhydrous DCM (5 mL). The mixture was stirred atambient temperature for ˜5 minutes to a clear solution. Intermediate 23(781 mg, 2.19 mmol) was then added and the mixture was stirred at roomtemperature overnight. Next day, diluted with DCM (50 mL) and washedwith H₂O (50 mL) followed by 10% K₂CO₃ (50 mL). Back-extraction wasperformed for both aqueous washes with DCM (2×40 mL). The organic layerswere combined, dried with MgSO₄, filtered, and concentrated in vacuo.The crude was purified by flash chromatography purification system (80 gsilica gel column) using a gradient of DCM for 1 min, then 0-30%MeOH/DCM for 30 min under the flow rate at 60 mL/min. The productfractions were collected and concentrated to yield Compound F8 (225 mg,68% yield) as a clear liquid. ¹H nmr (400 MHz, CDCl₃) δ: 4.20-4.15 (2H,m, NCH₂CH₂O), 3.57-3.54 (2H, m, NCH₂CH₂O), 3.40-3.33 (4H, m, CONCH₂),2.42-2.21 (12H, m, CH₂CH₂CH & CH₂N(CH₃)₂), 1.80-1.71 (2H, m,NCH₂CH₂CH₂N), 1.70-1.58 (4H, m, COCH₂CH₂), 1.29-1.15 (20H, m, CH₂),0.93-0.86 (6H, m, CH₃).

Compound F9: Compound F8 (620 mg, 1.09 mmol) was dissolved inacetonitrile (9 mL) and methyl iodide (1 mL) was added. The vial wasflushed with argon gas and allowed to stir at 40° C. for 4 hours.Afterwards, the mixture was concentrated in vacuo and purified by flashchromatography with loading the crude oil directly onto 24 g silica gelcolumn and using a gradient of DCM for 1 min, then 0-25% MeOH/DCM for 25min under the flow rate at 32 mL/min. The product fractions werecombined, concentrated, and subjected to Amberlyst A26 Anion Exchangeresin to yield Compound F9 (325 mg, 48% yield) as a clear oil. ¹H nmr(400 MHz, CDCl₃) δ: 4.21-4.19 (2H, m, NCH₂CH₂O), 3.86-3.81 (2H, m,NCH₂CH₂O), 3.47-3.44 (2H, m, CONCH₂), 3.41 (9H, s, N(CH₃)₃), 2.38-2.35(2H, m, CH₂N(CH₃)₃), 2.28-2.25 (2H, m, NCH₂CH₂CH₂N), 2.13-2.08 (2H, m,NCOCH₂CH₂N), 1.58-1.56 (4H, m, COCH₂CH₂), 1.29-1.17 (44H, m, CH₂),0.87-0.85 (6H, m, CH₃).

Example 20

A scheme for the preparation of Compounds C25 and C24 is shown in FIG.20.

Intermediate 24: To Fmoc-Dap(Boc).H₂O (10.0 g, 22.50 mmol) in anoven-dried flask (250 mL) with a magnetic bar were added anhydrous DCM(100 mL), linoleic acid (7.7 mL, 24.79 mmol), EDC (6.5 g, 33.80 mmol),and DMAP (0.6 g, 4.52 mmol) in sequence. The mixture was stirred atambient temperature stirred overnight (17 hours). The reaction mixturewas concentrated under reduced pressure, dissolved in 5 mL DCM andpurified by flash chromatography purification system (220 g silica gelcolumn) using a gradient of 0-50% EtOAc/hexane for 30 min under the flowrate at 60 mL/min. The product fractions were collected and concentratedto yield Intermediate 24 (15.2 g, 100% yield) as a clear liquid. Theproduct was verified with LC-MS prior to executing the next step—m/z of[M+H]⁺=675.98.

Intermediate 25: To intermediate 24 (3.0 g, 4.44 mmol) in an oven-driedflask (100 mL) with a magnetic bar were added anhydrous acetonitrile (20mL) and piperidine (2 mL). The mixture was stirred at ambienttemperature for 2 hours. The reaction mixture was concentrated,dissolved in 2 mL DCM and purified with a 40 g silica column using agradient of 0-50% EtOAc/hexane for 10 min, 0-15% MeOH/DCM for 20 minunder the flow rate at 40 mL/min. The product fractions were collectedand concentrated to yield Intermediate 25 (1.3 g, 65% yield) as a clearyellow liquid. The product was verified with LC-MS prior to executingthe next step—m/z of [M+H]⁺=453.82.

Intermediate 26: To intermediate 25 (1.3 g, 2.87 mmol) in an oven-driedflask (100 mL) with a magnetic bar were added anhydrous DCM (30 mL),linoleic acid (0.9 mL, 2.87 mmol), EDC (0.7 g, 3.44 mmol), and DMAP (0.1g, 0.57 mmol) in sequence. The mixture was stirred at ambienttemperature stirred overnight (17 hours). The reaction mixture was thenconcentrated under reduced pressure, dissolved in 2 mL DCM, and purifiedby flash chromatography purification system (24 g silica gel column)using a gradient of 0-20% EtOAc/hexane for 30 min under the flow rate at25 mL/min. The product fractions were collected and concentrated toyield Intermediate 26 (2.0 g, 97% yield) as a clear liquid. The productwas verified with LC-MS prior to executing the next step—m/z of[M+H]⁺=716.16.

Compound C25: To Intermediate 26 (2.0 g, 2.80 mmol) in an oven-driedflask (100 mL) with a magnetic bar were added anhydrous DCM (20 mL) andTFA (4 mL) in sequence. The mixture was stirred at ambient temperaturestirred for 2 hours. During stirring, the solution color turned fromclear to red. Next, the reaction mixture was first concentrated using arotavapor and washed with 10% K₂CO₃ aqueous solution (50 mL). Themixture was then extracted with DCM (2×50 mL) and the organic layerswere combined, dried over Na₂SO₄ (5 g), filtered, and concentrated underreduced pressure to give an oily residue, which was dissolved inanhydrous DCM (20 mL) and added DMA-Gly-OH.HCl (0.5 g, 3.37 mmol),EDC.HCL (0.8 g, 4.22 mmol), DMAP (0.2 g, 1.64 mmol), and TEA (0.2 mL,1.50 mmol) in sequence. The mixture was stirred at ambient temperaturefor 2.5 hours. Next, the reaction mixture was washed with 10% aqueousK₂CO₃ solution (50 mL), extracted with DCM (2×50 mL), dried over Na₂SO₄and concentrated by rotavapor. The crude was dissolved in 2 mL DCM andpurified with a 40 g silica column using a gradient of 0-15% MeOH/DCMfor 30 min under the flow rate at 40 mL/min. The product fractions werecollected and concentrated to yield Compound C25 (1.5 g, 76.6% yield) asa clear liquid. ¹H nmr (400 MHz, CDCl₃) δ: 7.50 (1H, bs, NH), 6.75 (1H,bs, NH), 5.33-5.35 (8H, m, CH═), 4.60-4.65 (1H, m, COCHN), 4.10-4.15(2H, m, OCH₂), 3.70-3.75 (2H, m, NCH₂), 2.90 (2H, s, NCH₂), 2.74-2.76(4H, m, ═CHCH₂CH═), 2.25 (6H, s, NCH₃), 2.23-2.24 (2H, m, CH₂CO),2.03-2.04 (8H, m, ═CHCH₂), 1.65-1.66 (4H, m, CH₂CH₂CO), 1.29-1.31 (30H,m, CH₂), 0.86-0.88 (6H, m, CH₃).

Compound C24: Compound C25 (1.5 g, 2.14 mmol) was added to methyl iodide(2 mL) in an oven-dried vial (20 mL) with a magnetic bar. The mixturewas stirred at ambient temperature overnight (17 hours). Next, theexcess reagent was removed under vacuum. The crude was dissolved in 1 mLDCM and purified with a 40 g silica column using a gradient of 0-15%MeOH/DCM for 30 min under the flow rate at 30 mL/min. The productfractions were combined, concentrated, and subjected to Amberlyst A26Anion Exchange resin to yield Compound C24 (1.6 g, 96% yield) as a clearliquid. ¹H nmr (400 MHz, CDCl₃) δ: 8.80 (1H, bs, NH), 6.90 (1H, bs, NH),5.33-5.35 (8H, m, CH═), 4.80-4.85 (2H, m, COCH₂N), 4.25-4.26 (1H, m,COCHN), 4.10-4.18 (2H, m, OCH₂), 3.80-3.81 (1H, m, NCH₂), 3.60-3.61 (1H,m, NCH₂), 3.45 (9H, s, NCH₃), 2.74-2.76 (4H, m, ═CHCH₂CH═), 2.37-2.38(2H, m, CH₂CO), 2.03-2.04 (8H, m, ═CHCH₂), 1.65-1.66 (4H, m, CH₂CH₂CO),1.29-1.31 (30H, m, CH₂), 0.86-0.88 (6H, m, CH₃).

Example 21

A scheme for the preparation of Compound D16 is shown in FIG. 21.

Intermediate 27: To intermediate 26 (10.3 g, 14.44 mmol) in anoven-dried flask (100 mL) with a magnetic bar were added anhydrous DCM(20 mL) and TFA (4 mL) in sequence. The mixture was stirred at ambienttemperature stirred for 2 hours. During stirring, the solution colorturned from clear to red. Next, the reaction mixture was firstconcentrated using a rotavapor and washed with 10% K₂CO₃ aqueoussolution (50 mL). The mixture was then extracted with DCM (2×50 mL) andthe organic layers were combined, dried over Na₂SO₄ (5 g), filtered, andconcentrated under reduced pressure to give an oily residue, which wasthen dissolved in anhydrous DCM (30 mL) and added bromoacetyl bromide(1.3 mL, 14.44 mmol), and TEA (2.2 mL, 15.85 mmol) slowly in sequence.The mixture was stirred at ambient temperature for 2.5 hours. Next, thereaction mixture was concentrated by rotavapor. The crude was dissolvedin 10 mL DCM and purified with a 220 g silica column using a gradient of0-50% EtOAc/hexane for 30 min under the flow rate at 60 mL/min. Theproduct fractions were collected and concentrated to yield Intermediate27 (8.0 g, 76% yield) as a clear liquid. The product was verified withLC-MS prior to executing the next step—m/z of [M+H]⁺=736.03.

Compound D16: Intermediate 27 (1.0 g, 1.36 mmol) was added to anhydrousDCM (20 mL) with DMSO (1 mL) in an oven-dried vial (40 mL) with amagnetic bar. Then were added cis-pyrrolidine-3,4-diol hydrochloride(cDHP.HCL) (0.3 g, 2.03 mmol) and TEA (0.5 mL, 3.41 mmol). The mixturewas stirred at ambient temperature for 2 hours. After removed thesolvent by rotavapor under vacuum, the residue was treated with 10%K₂CO₃ solution (50 mL) and extracted with DCM (2×50 mL). The organiclayers were then combined, dried over Na₂SO₄ (10 g), filtered, andconcentrated under reduced pressure. The crude was dissolved in 2 mL DCMand purified with a 24 g silica column using a gradient of 0-15%MeOH/DCM for 30 min under the flow rate at 25 mL/min. The productfractions were collected and concentrated to yield Compound D16 (0.8 g,78% yield) as a yellow liquid. ¹H nmr (400 MHz, CDCl₃) δ: 8.264 (1H, bs,NH), 6.71 (1H, d, J=5.2 Hz, NH), 5.34-5.37 (8H, m, CH═), 4.60-4.70 (1H,m, COCHN), 4.15-4.24 (4H, m, OCH₂, OCH), 3.83-3.86 (1H, m, COCH₂N),3.36-3.40 (1H, m, COCH₂N), 3.16 (2H, bs, COCH₂N), 3.09 (1H, d, J=8.4 Hz,NCH₂), 2.92 (1H, d, J=8.4 Hz, NCH₂), 2.77-2.93 (4H, m, ═CHCH₂CH═), 2.61(2H, bs, NCH₂), 2.28-2.31 (2H, m, CH₂CO), 2.03-2.07 (8H, m, ═CHCH₂),1.64-1.67 (4H, m, CH₂CH₂CO), 1.30-1.37 (30H, m, CH₂), 0.87-0.90 (6H, m,CH₃).

Example 22

A scheme for the preparation of Compound D17 is shown in FIG. 22.

Compound D17: Intermediate 27 (1.0 g, 1.36 mmol) was added to anhydrousDCM (20 mL) with DMSO (1 mL) in an oven-dried vial (40 mL) with amagnetic bar. Then were added trans-pyrrolidine-3,4-diol hydrochloride(tDHP.HCL) (0.3 g, 2.03 mmol), and TEA (0.5 mL, 3.41 mmol). The mixturewas stirred at ambient temperature for 2 hours. After removed thesolvent by rotavapor under vacuum, the residue was treated with 10%K₂CO₃ solution (50 mL) and extracted with DCM (2×50 mL). The organiclayers were then combined, dried over Na₂SO₄ (10 g), filtered, andconcentrated under reduced pressure. The crude was dissolved in 2 mL DCMand purified with a 24 g silica column using a gradient of 0-15%MeOH/DCM for 30 min under the flow rate at 25 mL/min. The productfractions were collected and concentrated to yield Compound D17 (1.1 g,100% yield) as a yellow liquid. ¹H nmr (400 MHz, CDCl₃) δ: 8.23 (1H, bs,NH), 6.68 (1H, d, J=5.2 Hz, NH), 5.30-5.37 (8H, m, CH═), 4.64-4.67 (1H,m, COCHN), 4.12-4.18 (4H, m, OCH₂, OCH), 3.91-3.94 (1H, m, COCH₂N),3.17-3.31 (3H, m, COCH₂N), 3.09-3.12 (2H, m, CH₂OH), 2.76-2.78 (4H, m,═CHCH₂CH═), 2.67-2.71 (2H, m, CH₂OH), 2.25-2.28 (2H, m, CH₂CO),2.03-2.07 (8H, m, ═CHCH₂), 1.63-1.77 (4H, m, OCH₂CH₂), CH₂CH₂CO),1.27-1.37 (30H, m, CH₂), 0.87-0.90 (6H, m, CH₃).

Example 23

A scheme for the preparation of Compound D18 is shown in FIG. 23.

Compound D18: Intermediate 27 (1.0 g, 1.36 mmol) was added to anhydrousDCM (20 mL) in an oven-dried vial (40 mL) with a magnetic bar. Then wereadded 1-(2-hydroxy)ethylpiperazine (0.2 mL, 1.63 mmol) and TEA (0.2 mL,1.5 mmol). The mixture was stirred at ambient temperature for 2 hours.After removed the solvent by rotavapor under vacuum, the residue wastreated with 10% K₂CO₃ solution (50 mL) and extracted with DCM (2×50mL). The organic layers were then combined, dried over Na₂SO₄ (10 g),filtered, and concentrated under reduced pressure. The crude wasdissolved in 2 mL DCM and purified with a 24 g silica column using agradient of 0-15% MeOH/DCM for 30 min under the flow rate at 25 mL/min.The product fractions were collected and concentrated to yield CompoundD18 (0.9 g, 84% yield) as a yellow liquid. ¹H nmr (400 MHz, CDCl₃) δ:7.48-7.50 (1H, m, NH), 6.68-6.70 (1H, s, NH), 5.29-5.35 (8H, m, CH═),4.59-4.62 (1H, m, COCHN), 4.10-4.12 (2H, m, OCH₂), 3.59-3.66 (4H, m,CH₂OH, COCH₂N), 2.99 (2H, s, COCH₂N), 2.75-2.76 (4H, m, ═CHCH₂CH═),2.53-2.55 (10H, m, CH₂N), 2.20-2.21 (2H, m, CH₂CO), 2.03-2.05 (8H, m,═CH—CH₂), 1.61-1.63 (4H, m, OCH₂CH₂), CH₂CH₂CO), 1.29-1.30 (30H, m,CH₂), 0.86-0.89 (6H, m, CH₃).

Example 24

A scheme for the preparation of Compound D19 is shown in FIG. 24.

Compound D19: Intermediate 27 (1.0 g, 1.36 mmol) was added to anhydrousDCM (20 mL) in an oven-dried vial (40 mL) with a magnetic bar. Then wereadded 1-(2-hydroxy)propylpiperazine (HPPip) (0.2 mL, 1.63 mmol) and TEA(0.2 mL, 1.50 mmol). The mixture was stirred at ambient temperature for2 hours. After removed the solvent by rotavapor under vacuum, theresidue was treated with 10% K₂CO₃ solution (50 mL) and extracted withDCM (2×50 mL). The organic layers were then combined, dried over Na₂SO₄(10 g), filtered, and concentrated under reduced pressure. The crude wasdissolved in 2 mL DCM and purified with a 24 g silica column using agradient of 0-15% MeOH/DCM for 30 min under the flow rate at 25 mL/min.The product fractions were collected and concentrated to yield CompoundD19 (0.9 g, 84% yield) as a yellow liquid. ¹H nmr (400 MHz, CDCl₃) δ:7.48-7.50 (1H, m, NH), 6.68-6.70 (1H, s, NH), 5.29-5.35 (8H, m, CH═),4.59-4.62 (1H, m, COCHN), 4.10-4.12 (2H, m, OCH₂), 3.59-3.66 (4H, m,CH₂OH, COCH₂N), 2.99 (2H, s, COCH₂N), 2.75-2.76 (4H, m, ═CHCH₂CH═),2.53-2.55 (10H, m, CH₂N), 2.20-2.21 (2H, m, CH₂CO), 2.03-2.05 (8H, m,═CHCH₂), 1.61-1.63 (4H, m, OCH₂CH₂), CH₂CH₂CO), 1.29-1.30 (30H, m, CH₂),0.86-0.89 (6H, m, CH₃).

Example 25

A scheme for the preparation of Compound D20 is shown in FIG. 25.

Intermediate 28: To N,N-dimethylglycine hydrochloride (0.6 g, 3.94 mmol)in an oven-dried flask (100 mL) with a magnetic bar were added anhydrousDCM (20 mL), H-Dap(Boc)-OMe.HCl (1.0 g, 3.94 mmol), EDC (1.0 g, 4.73mmol), and DMAP (0.1 g, 0.80 mmol) in sequence. The mixture was stirredat ambient temperature stirred overnight (17 hours). The reactionmixture was concentrated under reduced pressure and purified by flashchromatography purification system (24 g silica gel column) using agradient of 2-20% MeOH/DCM for 30 min under the flow rate at 25 mL/min.The product fractions were collected and concentrated to yieldIntermediate 28 (1.0 g, 84% yield) as a white solid. The product wasverified with LC-MS prior to executing the next step—m/z of[M+H]⁺=304.51.

Intermediate 29: To Intermediate 28 (1.0 g, 3.30 mmol) in an oven-driedvial (40 mL) with a magnetic bar were added methanol (10 mL) and LiOH(87 mg, 3.60 mmol). The mixture was stirred at ambient temperatureovernight (17 hours). The reaction mixture was concentrated to delivercrude Intermediate 29 for next step without purification. The productwas verified with LC-MS prior to executing the next step—m/z of[M+H]⁺=209.44.

Intermediate 30: To crude Intermediate 29 (˜1.1 g, ˜2.87 mmol) in anoven-dried flask (100 mL) with a magnetic bar were added anhydrous DCM(20 mL), linoleic acid (1.1 mL, 3.60 mmol), EDC (0.9 g, 4.80 mmol), andDMAP (0.1 g, 0.64 mmol) in sequence. The mixture was stirred at ambienttemperature stirred overnight (17 hours). The reaction mixture wasconcentrated under reduced pressure and purified by flash chromatographypurification system (24 g silica gel column) using a gradient of 0-20%MeOH/DCM for 30 min under the flow rate at 25 mL/min. The productfractions were collected and concentrated to yield Intermediate 30 (22mg, 13% yield) as a clear liquid. The product was verified with LC-MSprior to executing the next step—m/z of [M+H]+=538.84.

Compound D20: To Intermediate 30 (220 mg, 0.41 mmol) in an oven-driedflask (100 mL) with a magnetic bar were added anhydrous DCM (15 mL) andTFA (1 mL) in sequence. The mixture was stirred at ambient temperaturestirred for 2 hours. During stirring, the solution color turned fromclear to red. Next, the reaction mixture was first concentrated using arotavapor and washed with 10% K₂CO₃ aqueous solution (50 mL). Themixture was then extracted with DCM (2×50 mL) and the organic layerswere combined, dried over Na₂SO₄ (5 g), filtered, and concentrated underreduced pressure to give an oily residue, which was then dissolved inanhydrous DCM (30 mL) and added anhydrous DCM (20 mL), linoleic acid(0.2 mL, 0.49 mmol), EDC (118 mg, 0.62 mmol), and DMAP (10 mg, 0.08mmol) in sequence. The mixture was stirred at ambient temperaturestirred for 4 hours. The reaction mixture was concentrated using arotavapor and washed with 10% K₂CO₃ aqueous solution (50 mL). Themixture was then extracted with DCM (2×50 mL) and the organic layerswere combined, dried over Na₂SO₄ (5 g), filtered, and concentrated underreduced pressure. The crude was finally purified with a 12 g silicacolumn using a gradient of 0-15% MeOH/DCM for 30 min under the flow rateat 20 mL/min. The product fractions were collected and concentrated toyield Compound D20 (140 mg, 49% yield) as a clear liquid. ¹H nmr (400MHz, CDCl₃) δ: 7.90 (1H, bs, NH), 6.10 (1H, bs, NH), 5.60-5.70 (8H, m,CH═), 4.65 (1H, bs, COCHN), 4.20-4.25 (2H, m, OCH₂), 3.58-3.71 (2H, m,CH₂N), 2.90-2.91 (2H, m, COCH₂N), 2.75-2.76 (4H, m, ═CHCH₂CH₂), 2.30(6H, s, NCH₃), 2.10-2.11 (2H, m, COCH₂), 2.0-2.1 (8H, m, ═CHCH₂),1.54-1.69 (4H, m, OCH₂CH₂), CH₂CH₂CO), 1.10-1.30 (30H, m, CH₂),0.80-0.82 (6H, m, CH₃).

Example 26

A scheme for the preparation of Compound D21 is shown in FIG. 26.

Compound D21: To(2R)-3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyldioleate (450 mg, 0.58 mmol) in an oven-dried vial (40 mL) with amagnetic bar were added anhydrous DCM (8 mL), 2-Methoxyethoxymethylchloride (133 mL, 1.16 mmol) and potassium carbonate (332 mg, 2.32 mmol)in sequence. The mixture was stirred at ambient temperature for oneweek. The reaction mixture was filtered, and concentrated under reducedpressure, which was purified with a 12 g silica column using a gradientof 0-20% MeOH/DCM for 30 min under the flow rate at 20 mL/min. Theproduct fractions were collected and concentrated to yield Compound D21(150 mg, 30% yield) as a clear liquid. ¹H nmr (400 MHz, CDCl₃) δ:75.29-5.33 (8H, m, CH═), 5.20 (1H, bs, OCH), 4.92 (2H, s, OCH₂O),4.31-4.40 (2H, m, OCH₂), 3.98-4.04 (4H, m, OCH₂), 3.70-3.71 (2H, m,OCH₂), 3.56-3.57 (2H, m, OCH₂), 3.36 (3H, s, OCH₃), 3.24 (6H, s, NCH₃),2.27-2.90 (2H, m, COCH₂), 1.99-2.00 (8H, m, ═CHCH₂), 1.54-1.69 (4H, m,COCH₂CH₂), 1.20-1.30 (40H, m, CH₂), 0.85-0.88 (6H, m, CH₃).

Example 27

A scheme for the preparation of Compound E37 is shown in FIG. 27.

Intermediate 31: To 1-(tert-butyl)-2-methyl(2S,4R)-4-aminopyrrolidine-1,2-dicarboxylate hydrochloride (2.0 g, 7.12mmol) in an oven-dried flask (100 mL) with a magnetic bar were addedanhydrous DCM (25 mL), 3-(dimethylamino)-propanoic acid (1.3 g, 8.55mmol),1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid-hexafluorophosphate(4.1 g, 10.68 mmol), and DIEA (2.6 mL, 14.96 mmol) in sequence. Themixture was stirred at ambient temperature stirred overnight (17 hours).The reaction mixture was concentrated under reduced pressure andpurified by flash chromatography purification system (24 g silica gelcolumn) using a gradient of 0-10% MeOH/DCM for 30 min under the flowrate at 25 mL/min. The product fractions were collected and concentratedto yield Intermediate 31 (1.5 g, 85% yield) as a clear oil. The productwas verified with LC-MS prior to executing the next step—m/z of[M+H]⁺=344.57.

Intermediate 32: To Intermediate 31 (1.5 g, 4.25 mmol) in an oven-driedflask (50 mL) with a magnetic bar were added lithium hydroxide (1.2 mg,5.10 mmol), water (5 mL) and MeOH (5 mL). The mixture was stirred atambient temperature overnight. Next solvents were removed under reducedpressure and the wet material was dried via lyophilization to give acrude Intermediate 32 (1.3 g, 95% yield) as a white solid, which wasused without further purification. The crude was verified with LC-MSprior to executing the next step—m/z of [M+H]⁺=330.00.

Intermediate 33: To Intermediate 32 (1.3 g, 3.95 mmol) in an oven-driedflask (100 mL) with a magnetic bar were added anhydrous DMF (20 mL),linoleyl alcohol (1.4 g, 5.14 mmol), EDC (1.5 g, 7.90 mmol), and DMAP(0.5 g, 3.95 mmol) in sequence. The mixture was stirred at ambienttemperature stirred overnight (17 hours). The reaction mixture wasdiluted with DCM (25 mL), washed with sodium bicarbonate (20 mL), driedover Na₂SO₄ (5 g), filtered, and concentrated under reduced pressure.The crude was purified by flash chromatography purification system (24 gsilica gel column) using a gradient of 0-10% MeOH/DCM for 30 min underthe flow rate at 25 mL/min. The product fractions were collected andconcentrated to yield Intermediate 33 (1.9 g, 85% yield) as a clear oil.The product was verified with LC-MS prior to executing the next step—m/zof [M+H]⁺=578.91.

Intermediate 34: To Intermediate 33 (1.9 g, 3.33 mmol) in an oven-driedflask (25 mL) with a magnetic bar were added anhydrous DCM (10 mL) andTFA (2 mL) in sequence. The mixture was stirred at ambient temperaturestirred for 2 hours. During stirring, the solution color turned fromclear to red. Next, the reaction mixture was first concentrated using arotavapor and washed with 10% Na₂CO₃ solution (20 mL). The mixture wasthen extracted with DCM (2×20 mL) and the organic layers were combined,dried over Na₂SO₄ (5 g), filtered, and concentrated under reducedpressure to give an oily Intermediate 34 (1.5 g, 92% yield) which wasused for the next step without further purification. The product wasverified with LC-MS prior to executing the next step—m/z of[M+H]⁺=478.82.

Compound E37: To Intermediate 34 (1.5 g, 3.14 mmol) in an oven-driedflask (100 mL) with a magnetic bar were added anhydrous DMF (10 mL),linoleic acid (1.3 g, 4.71 mmol), and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid-hexafluorophosphate (2.3 g, 6.28 mmol) in sequence. The mixturewas stirred at ambient temperature stirred overnight (17 hours). Thereaction mixture was concentrated under reduced pressure and purified byflash chromatography purification system (24 g silica gel column) usinga gradient of 0-10% MeOH/DCM for 30 min under the flow rate at 25mL/min. The product fractions were collected and concentrated to yieldCompound E37 (2.0 g, 92% yield) as a clear oil. ¹H nmr (400 MHz, CDCl₃)δ: 8.0 (1H, bs, NH) 5.54-5.25 (8H, m, CH═), 4.65-4.29 (2H, m, NHCH,NCHCO), 4.30-3.90 (2H, m, OCH₂), 3.80-3.62 (2H, m, NCH₂CHNH—), 3.60-3.45(2H, m, (CH₃)₂NCH₂), 2.84 (6H, s, N(CH₃)₂), 2.80-2.60 (6H, m, CH₂),2.40-1.90 (12H, m, CH₂CONH, CH₂CH═), 1.65-1.48 (4H, m, CH₂), 1.45-1.20(30H, m, CH₂), 0.86-0.85 (6H, m, CH₃).

Example 28

A scheme for the preparation of Compounds E38 and E39 is shown in FIG.28.

Intermediate 35: To 1-(tert-butyl)-2-methyl(2S,4R)-4-aminopyrrolidine-1,2-dicarboxylate hydrochloride (5.0 g, 17.81mmol) in an oven-dried flask (100 mL) with a magnetic bar were addedanhydrous DCM (25 mL), 3-(dimethylamino)butanoic acid (3.6 g, 21.37mmol), EDC (6.8 g, 35.62 mmol), DMAP (2.2 g, 17.81 mmol), and DIEA (8.1g, 62.34 mmol) in sequence. The mixture was stirred at ambienttemperature stirred overnight (17 hours). The reaction mixture was thendiluted with DCM (25 mL), washed with sodium bicarbonate (20 mL), driedover Na₂SO₄ (5 g), filtered, and concentrated under reduced pressure.The crude was purified by flash chromatography purification system (120g silica gel column) using a gradient of 0-10% MeOH/DCM for 30 min underthe flow rate at 25 mL/min. The product fractions were collected andconcentrated to yield Intermediate 35 (6.1 g, 80% yield) as a clear oil.The product was verified with LC-MS prior to executing the next step—m/zof [M+H]⁺=358.59.

Intermediate 36: To Intermediate 35 (6.1 g, 17.03 mmol) in an oven-driedflask (50 mL) with a magnetic bar were added lithium hydroxide (0.5 g,20.44 mmol), water (5 mL) and MeOH (20 mL). The mixture was stirred atambient temperature overnight. Next solvents were removed under reducedpressure and the wet material was dried via lyophilization to give acrude Intermediate 36 (5.6 g, 95% yield) as a white solid, which wasused without further purification. The crude was verified with LC-MSprior to executing the next step—m/z of [M+H]⁺=344.55.

Intermediate 37: To Intermediate 36 (2.8 g, 8.11 mmol) in an oven-driedflask (100 mL) with a magnetic bar were added anhydrous DMF (10 mL),linoley alcohol (2.6 g, 9.73 mmol), and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid-hexafluorophosphate (4.6 g, 12.16 mmol) in sequence. The mixturewas stirred at ambient temperature stirred overnight (17 hours). Thereaction mixture was diluted with DCM (25 mL), washed with sodiumbicarbonate (20 mL) dried over Na₂SO₄ (5 g), filtered, and concentratedunder reduced pressure. The crude was purified by flash chromatographypurification system (24 g silica gel column) using a gradient of 0-10%MeOH/DCM for 30 min under the flow rate at 25 mL/min. The productfractions were collected and concentrated to yield Intermediate 37 (3.9g, 82% yield) as a white solid. The product was verified with LC-MSprior to executing the next step—m/z of [M+H]⁺=592.91.

Intermediate 38: To Intermediate 37 (1.4 g, 2.31 mmol) in an oven-driedflask (25 mL) with a magnetic bar were added anhydrous DCM (20 mL) andTFA (5 mL) in sequence. The mixture was stirred at ambient temperaturestirred for 2 hours. During stirring, the solution color turned fromclear to red. Next, the reaction mixture was first concentrated using arotavapor and washed with 10% Na₂CO₃ solution (20 mL). The mixture wasthen extracted with DCM (2×20 mL) and the organic layers were combined,dried over Na₂SO₄ (5 g), filtered, and concentrated under reducedpressure to give a light yellow oily Intermediate 38 (580 mg, 51% yield)which was used for the next step without further purification. Theproduct was verified with LC-MS prior to executing the next step—m/z of[M+H]⁺=492.81.

Compound E38: To Intermediate 38 (580 mg, 1.18 mmol) in an oven-driedflask (100 mL) with a magnetic bar were added anhydrous DMF (5 mL),linoleic acid (460 mg, 1.53 mmol), and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid-hexafluorophosphate (898 mg, 2.36 mmol) in sequence. The mixturewas stirred at ambient temperature stirred overnight (17 hours). Thereaction mixture was concentrated under reduced pressure and purified byflash chromatography purification system (24 g silica gel column) usinga gradient of 0-10% MeOH/DCM for 30 min under the flow rate at 25mL/min. The product fractions were collected and concentrated to yieldCompound E38 (750 mg, 95% yield) as a clear oil. ¹H nmr (400 MHz, CDCl₃)δ: 5.45-5.25 (8H, m, CH═), 4.70-4.29 (2H, m, NHCH, NCHCO), 4.20-4.00(2H, m, OCH₂), 3.90-3.78 (2H, m, NCH₂CHNH—), 3.75-3.62 (2H, m, Me₂NCH₂),2.84 (6H, s, N (CH₃)₂), 2.90-2.70 (4H, m, CH₂), 2.60-2.55 (2H, m, CH₂),2.10-1.90 (12H, m, CH₂CONH, CH₂CH═), 1.65-1.40 (6H, m, CH₂), 1.40-1.20(30H, m, CH₂), 0.90-0.80 (6H, m, CH₃).

Compound E39: Compound E38 (350 mg, 0.46 mmol) was added to methyliodide (2 mL) in an oven-dried vial (20 mL) with a magnetic bar. Themixture was stirred at ambient temperature overnight (17 hours). Next,the excess reagent was removed under vacuum. The crude was dissolved in1 mL DCM and purified with a 40 g silica column using a gradient of0-15% MeOH/DCM for 30 min under the flow rate at 30 mL/min. The productfractions were combined, concentrated, and subjected to Amberlyst A26Anion Exchange resin to yield Compound E39 (165 mg, 82% yield) as aclear liquid. ¹H nmr (400 MHz, CDCl₃) δ: 5.45-5.25 (8H, m, CH═),4.70-4.35 (2H, m, NHCH, NCHCO), 4.20-4.00 (2H, m, OCH₂), 3.90-3.40 (4H,m, NCH₂CH₂CH₂CO—, NCH₂CHNH—), 3.30 (9H, s, N⁺ (CH₃)₃), 2.85-2.70 (4H, m,CH₂), 2.60-2.45 (2H, m, CH₂), 2.45-2.10 (8H, m, CH₂CH₂CH═), 2.10-2.00(6H, m, CH₂), 1.70-1.40 (4H, m, CH₂), 1.40-1.20 (30H, m, CH₂), 0.90-0.70(6H, m, CH₃).

Example 29

A scheme for the preparation of Compound E40 is shown in FIG. 29.

Intermediate 39: To(2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid(5.0 g, 21.62 mmol) in an oven-dried flask (100 mL) with a magnetic barwere added anhydrous acetonitrile (500 mL), TBDPS-Cl (11.9 g, 43.24mmol) and 1,8-diazabicycloundec-7-ene (11.5 g, 75.67 mmol) in sequence.The mixture was refluxed at 50° C. for 4 hours. Then, the solvent wasremoved under reduced pressure. The residue was dissolved in 0.2 N HCl(50 mL), extracted with DCM (2×50 mL), dried over Na₂SO₄ (5 g),filtered, and concentrated under reduced pressure. The crude waspurified by flash chromatography purification system (330 g silica gelcolumn) using a gradient of 0-5% MeOH/DCM for 30 min under the flow rateat 25 mL/min. The product fractions were collected and concentrated toyield Intermediate 39 (9.9 g, 97% yield) as a clear oil. The product wasverified with LC-MS prior to executing the next step—m/z of[M+H]⁺=470.49.

Intermediate 40: To Intermediate 39 (9.9 g, 21.29 mmol) in an oven-driedflask (250 mL) with a magnetic bar were added anhydrous DCM (100 mL),linoleyl alcohol (6.8 g, 25.55 mmol), EDC (8.2 g, 42.58 mmol), and DMAP(1.3 g, 10.65 mmol) in sequence. The mixture was stirred at ambienttemperature stirred overnight (17 hours). The reaction mixture wasdiluted with DCM (25 mL), washed with sodium bicarbonate (20 mL) driedover Na₂SO₄ (5 g), filtered, and concentrated under reduced pressure.The crude was purified by flash chromatography purification system (330g silica gel column) using a gradient of 0-20% MeOH/DCM for 30 min underthe flow rate at 25 mL/min. The product fractions were collected andconcentrated to yield Intermediate 40 (12.8 g, 98% yield) as a whitesolid. The product was verified with LC-MS prior to executing the nextstep—m/z of [M+H]⁺=718.75.

Intermediate 41: To Intermediate 40 (5.8 g, 6.96 mmol) in an oven-driedflask (50 mL) with a magnetic bar were added 4 N HCl in 1, 4-dioxanesolution (20 mL). The mixture was then stirred at ambient temperaturefor 3 hours. Next, the reaction mixture was concentrated under reducedpressure to gain a clear oil, which was then dissolved in anhydrous DCM(20 mL) and added linoleic acid (2.3 g, 8.36 mmol), EDC (2.0 g, 10.45mmol), and DMAP (0.6 g, 4.87 mmol) in sequence. The mixture was stirredat ambient temperature stirred overnight (17 hours). The reactionmixture was diluted with DCM (25 mL), washed with sodium bicarbonate (20mL) dried over Na₂SO₄ (5 g), filtered, and concentrated under reducedpressure. The crude was purified by flash chromatography purificationsystem (330 g silica gel column) using a gradient of 0-20% MeOH/DCM for30 min under the flow rate at 25 mL/min. The product fractions werecollected and concentrated to yield Intermediate 41 (5.8 g, 94% yield)as a colorless oil. The product was verified with LC-MS prior toexecuting the next step—m/z of [M+H]⁺=880.91.

Intermediate 42: To Intermediate 41 (5.8 g, 6.58 mmol) in an oven-driedflask (250 mL) with a magnetic bar were added THF (100 mL) andtriethylamine trihydrofluoride (10.6 g, 65.8 mmol) sequentially. Themixture was then stirred at ambient temperature for 17 hours. Next, thereaction mixture was concentrated under reduced pressure and purified byflash chromatography purification system (120 g silica gel column) usinga gradient of 0-30% EtOAc/hexane for 10 min then 0-10% MeOH/DCM for 20min under the flow rate at 25 mL/min. The product fractions werecollected and concentrated to yield Intermediate 42 (4.3 g, 78% yield)as a colorless oil. The product was verified with LC-MS prior toexecuting the next step—m/z of [M+H]⁺=642.91.

Intermediate 43: To Intermediate 42 (2.3 g, 7.16 mmol) in an oven-driedflask (50 mL) with a magnetic bar were added DCM (10 mL), 2-bromoacetylbromide (0.9 g, 4.66 mmol) and TEA (0.7 g, 7.16 mmol) sequentially. Themixture was then stirred at ambient temperature for 3 hours. Next, thereaction mixture was concentrated under reduced pressure and purified byflash chromatography purification system (120 g silica gel column) usinga gradient of 0-30% EtOAc/hexane for 10 min then 0-10% MeOH/DCM for 20min under the flow rate at 25 mL/min. The product fractions werecollected and concentrated to yield Intermediate 43 (2.4 g, 82% yield)as a colorless oil. The product was verified with LC-MS prior toexecuting the next step—m/z of [M+H]⁺=764.74.

Compound E40: Intermediate 43 (300 mg, 0.39 mmol) was added to anhydrousDCM (5 mL) in an oven-dried vial (40 mL) with a magnetic bar. Then3-azetidinemethanol hydrochloride (97 mg, 0.79 mmol) and TEA (0.2 mL,1.18 mmol) were added in sequence. The mixture was stirred at ambienttemperature for 17 hours. After removed the solvent by rotavapor undervacuum, the residue was treated with 10% K₂CO₃ solution (50 mL) andextracted with DCM (2×50 mL). The organic layers were then combined,dried over Na₂SO₄ (10 g), filtered, and concentrated under reducedpressure. The crude was dissolved in 2 mL DCM and purified with a 24 gsilica column using a gradient of 0-15% MeOH/DCM for 30 min under theflow rate at 25 mL/min. The product fractions were collected andconcentrated to yield Compound E40 (196 mg, 65% yield) as a yellowliquid. The lipid's identity was confirmed with LC-MS analysis—m/z of[M+H]⁺=769.17.

Example 30

A scheme for the preparation of Compound A23 is shown in FIG. 30.

Intermediate 44: To O-benzoyl-N-(tert-butoxycarbonyl)-L-serine (3.0 g,10.16 mmol) in an oven-dried flask (100 mL) with a magnetic bar wereadded anhydrous DCM (40 mL), myristic amine (2.4 g, 11.17 mmol), EDC(2.9 g, 15.24 mmol), and DMAP (0.6 g, 5.08 mmol) in sequence. Themixture was stirred at ambient temperature stirred overnight (17 hours).The reaction mixture was diluted with EtOAc (300 mL), washed with sodiumbicarbonate (20 mL) dried over Na₂SO₄ (5 g), filtered, and concentratedunder reduced pressure. The crude was purified by flash chromatographypurification system (330 g silica gel column) using 1:2 v/v EtOAc/hexanefor 30 min under the flow rate at 25 mL/min. The product fractions werecollected and concentrated to yield Intermediate 44 (4.4 g, 89% yield)as a white solid. The product was verified with LC-MS prior to executingthe next step—m/z of [M+H]⁺=491.61.

Intermediate 45: To Intermediate 44 (2.0 g, 4.08 mmol) in an oven-driedflask (50 mL) with a magnetic bar were added MeOH (30 mL) and palladiumon carbon (120 mg). The flask was then purge with hydrogen using ahydrogen balloon. The mixture was stirred at ambient temperature for 5hours and filtered via a bed of celite. The filtrate was thenconcentrated under reduced pressure to gain Intermediate 45 (1.7 g, 95%)as a white solid, which was used for the next step without furtherpurification. The product was verified with LC-MS prior to executing thenext step—m/z of [M+H]⁺=401.44.

Intermediate 46: To Intermediate 45 (1.7 g, 4.28 mmol) in an oven-driedflask (50 mL) with a magnetic bar were added anhydrous DCM (20 mL),myristic acid (1.3 g, 5.56 mmol), EDC (1.6 g, 8.55 mmol), and DMAP (0.5g, 4.28 mmol) in sequence. The mixture was stirred at ambienttemperature stirred overnight (17 hours). The reaction mixture wasdiluted with EtOAc (100 mL), washed with 1 N HCl (20 mL) dried overNa₂SO₄ (5 g), filtered, and concentrated under reduced pressure. Thecrude was purified by flash chromatography purification system (120 gsilica gel column) using 1:2 v/v EtOAc/hexane for 30 min under the flowrate at 25 mL/min. The product fractions were collected and concentratedto yield Intermediate 46 (2.4 g, 94% yield) as a white solid. Theproduct was verified with LC-MS prior to executing the next step—m/z of[M+H]⁺=612.02.

Compound A23: To Intermediate 46 (435 mg, 0.85 mmol) in an oven-driedflask (25 mL) with a magnetic bar were added anhydrous DCM (20 mL) andTFA (5 mL) in sequence. The mixture was stirred at ambient temperaturestirred for 2 hours. During stirring, the solution color turned fromclear to red. Next, the reaction mixture was first concentrated using arotavapor and washed with 10% Na₂CO₃ solution (20 mL). The mixture wasthen extracted with DCM (2×20 mL) and the organic layers were combined,dried over Na₂SO₄ (5 g), filtered, and concentrated to an oily residue,which was then in DCM (10 mL) and added succinic anhydride (118 mg, 1.17mmol), and TEA (118 mg, 1.17 mmol). The mixture was stirred at ambienttemperature stirred overnight (17 hours). The reaction mixture wasdiluted with DCM (100 mL), washed with sodium bicarbonate (20 mL) driedover Na₂SO₄ (5 g), filtered, and concentrated under reduced pressure.The crude was purified by flash chromatography purification system (120g silica gel column) using 0-20% MeOH/DCM for 30 min under the flow rateat 25 mL/min. The product fractions were collected and concentrated toyield Compound A23 (338 mg, 92% yield) as a white solid. ¹H nmr (400MHz, CDCl₃) δ: 6.76-6.70 (1H, m, CHNHCO), 6.40-6.35 (1H, m, CH₂NHCO),4.70-4.60 (1H, m, NHCHCO), 4.42-4.20 (2H, m, CH₂ 0), 3.26-3.12 (2H, m,NHCH₂), 2.80-2.60 (2H, m, CH₂CO₂H), 2.57-2.50 (2H, m, CH₂CONH),2.30-2.20 (2H, m, CH₂CO₂), 1.60-1.50 (2H, m, CH₂CH₂CO₂), 1.50-1.40 (2H,CH₂CH₂NH), 1.35-1.15 (42H, m, NH(CH₂)₂(CH₂)₁₁Me, CO(CH₂)₂(CH₂)₁₀Me,),0.90-0.83 (6H, m, CH₃).

Example 31

A scheme for the preparation of Compound A24 is shown in FIG. 31.

Intermediate 46: ToN-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-trityl-L-serine (3.0 g, 5.27mmol) in an oven-dried flask (100 mL) with a magnetic bar were addedanhydrous DCM (40 mL), linoleyl alcohol (1.5 g, 5.79 mmol), EDC (2.0 g,10.54 mmol), and DMAP (3.7 g, 0.70 mmol) in sequence. The mixture wasstirred at ambient temperature stirred overnight (17 hours). Thereaction mixture was diluted with DCM (100 mL), washed with sodiumbicarbonate (20 mL) dried over Na₂SO₄ (5 g), filtered, and concentratedunder reduced pressure. The crude was purified by flash chromatographypurification system (120 g silica gel column) using 1:2 v/v EtOAc/hexanefor 30 min under the flow rate at 25 mL/min. The product fractions werecollected and concentrated to yield Intermediate 46 (4.3 g, 90% yield)as a clear oil. The product was verified with LC-MS prior to executingthe next step—m/z of [M+NH₄]+=835.00.

Intermediate 47: To Intermediate 46 (4.3 g, 5.19 mmol) in an oven-driedflask (50 mL) with a magnetic bar were added 3 N HCl in MeOH solution(40 mL). The mixture was then stirred at ambient temperature for 3hours. Next, the reaction mixture was concentrated under reducedpressure to gain Intermediate 47 (2.5 g, 86% yield) as a clear oil,which was then used for the next reaction without further purification.The product was verified with LC-MS prior to executing the next step—m/zof [M+H]⁺=576.68.

Intermediate 48: To Intermediate 47 (2.5 g, 4.34 mmol) in an oven-driedflask (100 mL) with a magnetic bar were added anhydrous DCM (50 mL),linoleic acid (1.3 g, 4.78 mmol), EDC (1.7 g, 8.68 mmol), and DMAP (0.3g, 2.17 mmol) in sequence. The mixture was stirred at ambienttemperature stirred overnight (17 hours). The reaction mixture wasdiluted with DCM (100 mL), washed with 1 N sodium bicarbonate (20 mL)dried over Na₂SO₄ (5 g), filtered, and concentrated under reducedpressure. The crude was purified by flash chromatography purificationsystem (120 g silica gel column) using 1:2 v/v EtOAc/hexane for 30 minunder the flow rate at 25 mL/min. The product fractions were collectedand concentrated to yield Intermediate 48 (2.1 g, 82% yield) as a whitesolid. The product was verified with LC-MS prior to executing the nextstep—m/z of [M+NH₄]⁺=856.06.

Intermediate 49: To Intermediate 48 (2.9 g, 3.40 mmol) in an oven-driedflask (50 mL) with a magnetic bar were added anhydrous acetonitrile (20mL) and piperidine (4 mL). The mixture was stirred at ambienttemperature stirred overnight (17 hours). the reaction mixture wasconcentrated under reduced pressure and purified by flash chromatographypurification system (120 g silica gel column) using 0-50% EtOAc/hexanefor 30 min under the flow rate at 40 mL/min. The product fractions werecollected and concentrated to yield Intermediate 49 (1.3 g, 90% yield)as a clear oil. The product was verified with LC-MS prior to executingthe next step—m/z of [M+H]⁺=617.06.

Intermediate 50: Intermediate 49 (500 mg, 0.81 mmol) was dissolved inanhydrous DCM (10 mL) and bromoacetyl bromide (0.1 mL, 0.81 mmol)followed by TEA (0.1 mL, 0.89 mmol) were then added slowly. After theaddition was completed, the mixture was stirred at ambient temperatureovernight. Next day, the mixture was diluted with DCM (50 mL) and washedwith H₂O (50 mL) and 10% K₂CO₃ (50 mL). Back-extraction was performedfor both aqueous washes with DCM (2×25 mL). The organic layers werecombined, dried with MgSO₄, filtered, and concentrated in vacuo. Thecrude was purified with a 40 g silica column on flash chromatographysystem equipped with ESLD detector using a gradient of hexane for 0.5min, then 0-50% EtOAc/hexane 30 min gradient followed by 50%EtOAc/hexane for 5 min under the flow rate at 40 mL/min. The productfractions were collected and concentrated to yield Intermediate 50 (445mg, 80% yield) as a colorless liquid. The product was verified withLC-MS prior to executing the next step—m/z of [M+H]⁺=737.01.

Compound A24: Intermediate 50 (150 mg, 0.20 mmol) was added to anhydrousDCM (5 mL) in an oven-dried vial (40 mL) with a magnetic bar. Then wereadded 3-(piperazin-1-yl)propan-1-ol (33 mg, 0.22 mmol) and TEA (40 mL,0.25 mmol). The mixture was stirred at ambient temperature for 4 hours.After removed the solvent by rotavapor under vacuum, the residue wastreated with 10% K₂CO₃ solution (50 mL) and extracted with DCM (2×50mL). The organic layers were then combined, dried over Na₂SO₄ (10 g),filtered, and concentrated under reduced pressure. The crude wasdissolved in 2 mL DCM and purified with a 24 g silica column using agradient of 10-15% EtOAc/hexane for 10 min then 0-10% MeOH/DCM for 30min under the flow rate at 25 mL/min. The product fractions werecollected and concentrated to yield Compound A24 (128 mg, 70% yield) asa colorless liquid. ¹H nmr (400 MHz, CDCl₃) δ: 6.50-6.40 (1H, m, NHCO),5.45-5.20 (8H, m, CH═), 4.90-4.60 (2H, m, NHCHCO, HO) 4.60-4.50 (1H, m,CH₂CHOC(O)), 4.45-4.30 (1H, m, CH₂CHOC(O)), 4.20-4.10 (2H, m,C(O)OCH₂CH₂), 3.80-3.70 (2H, m, HOCH₂CH₂), 3.47 (2H, s, NCH₂C(O)NH),2.85-2.70 (4H, m, ═CHCH₂CH═), 2.70-2.45 (8H, m, HOCH₂CH₂CH₂N,CH₂NCH₂C(O), O(CO)CH₂CH₂), 2.30-1.90 (12H, m, NCH₂CH₂NCH₂C(O),CH₂CH₂CH═), 1.60-1.10 (36H, m, CH₂), 0.90-0.75 (6H, m, CH₃).

Example 32

A scheme for the preparation of Compound A25 is shown in FIG. 32.

Compound A25: Intermediate 50 (208 mg, 0.28 mmol) was added to anhydrousDCM (5 mL) in an oven-dried vial (40 mL) with a magnetic bar. Then3-azetidinemethanol hydrochloride (70 mg, 0.26 mmol) and TEA (120 mL,0.85 mmol) were added. The mixture was stirred at ambient temperaturefor 2 hours. After removed the solvent by rotavapor under vacuum, theresidue was treated with 10% K₂CO₃ solution (50 mL) and extracted withDCM (2×50 mL). The organic layers were then combined, dried over Na₂SO₄(10 g), filtered, and concentrated under reduced pressure. The crude wasdissolved in 2 mL DCM and purified with a 24 g silica column using agradient of 0-10% MeOH/DCM for 30 min under the flow rate at 25 mL/min.The product fractions were collected and concentrated to yield CompoundA25 (66 mg, 31% yield) as a colorless liquid. ¹H nmr (400 MHz, CDCl₃) δ:6.60-6.50 (1H, m, NHCO), 5.40-5.20 (8H, m, CH═), 4.90-4.80 (2H, m,CH₂CH₂OC(O)), 4.52-4.45 (1H, m, NHCHCO), 4.13-4.02 (2H, m, CH₂OC(O)),3.60-3.40 (2H, m, CH₂OH), 3.70-3.30 (4H, m, CHCH₂N), 3.20 (2H, s, CH₂N),2.80-2.65 (5H, m, ═CHCH₂CH═, —OH), 2.70-2.40 (3H, m, CH₂CH₂CO(O),HOCH₂CH), 2.30-1.90 (8H, m, CH₂CH₂CH═), 1.60-1.10 (34H, m, CH₂),0.90-0.83 (6H, m, CH₃).

Example 33

Example formulations are prepared for encapsulating a small interferingnucleic acid agents (siRNA), as shown in Table 3.

TABLE 3 Lipid formulations for siRNA Compound Choles- DPPE- C2 terolDOPE DOPC mPEG(2000) EE* No. (mol %) (mol %) (mol %) (mol %) (mol %) (%)1 25 30 30 10 5 >90 2 25 30 25 15 5 >90 3 25 30 20 20 5 >90 4 25 30 1525 5 >90 5 25 30 10 30 5 >90 6 25 35 15 20 5 >90 7 25 35 20 15 5 >90 830 30 15 20 5 >90 9 30 30 20 15 5 >90 10 35 30 15 15 5 >90*Encapsulation efficiency for siRNA.

Example 34

Example formulations are prepared for encapsulating a small interferingnucleic acid agents (siRNA), as shown in Table 4.

TABLE 4 Lipid formulations for siRNA Compound Choles- DPPE- A9 terolDOPE DOPC mPEG(2000) EE* No. (mol %) (mol %) (mol %) (mol %) (mol %) (%)1 25 30 30 10 5 >90 2 25 30 25 15 5 >90 3 25 30 20 20 5 >90 4 25 30 1525 5 >90 5 25 30 10 30 5 >90 6 25 35 15 20 5 >90 7 25 35 20 15 5 >90 830 30 15 20 5 >90 9 30 30 20 15 5 >90 10 35 30 15 15 5 >90*Encapsulation efficiency for siRNA.

Example 35

Example formulations are prepared for encapsulating a small interferingnucleic acid agents (siRNA), as shown in Table 5.

TABLE 5 Lipid formulations for siRNA Compound Choles- DPPE- AA terolDOPE DOPC mPEG(2000) EE* No. (mol %) (mol %) (mol %) (mol %) (mol %) (%)1 25 30 30 10 5 >90 2 25 30 25 15 5 >90 3 25 30 20 20 5 >90 4 25 30 1525 5 >90 5 25 30 10 30 5 >90 6 25 35 15 20 5 >90 7 25 35 20 15 5 >90 830 30 15 20 5 >90 9 30 30 20 15 5 >90 10 35 30 15 15 5 >90*Encapsulation efficiency for siRNA.

Example 36

Example formulations are prepared for encapsulating a small interferingnucleic acid agents (siRNA), as shown in Table 6.

TABLE 6 Lipid formulations for siRNA Compound Choles- DPPE- F5 terolDOPE DOPC mPEG(2000) EE* No. (mol %) (mol %) (mol %) (mol %) (mol %) (%)1 25 30 30 10 5 >90 2 25 30 25 15 5 >90 3 25 30 20 20 5 >90 4 25 30 1525 5 >90 5 25 30 10 30 5 >90 6 25 35 15 20 5 >90 7 25 35 20 15 5 >90 830 30 15 20 5 >90 9 30 30 20 15 5 >90 10 35 30 15 15 5 >90*Encapsulation efficiency for siRNA.

Example 37

Formulations were prepared for encapsulating a small interfering nucleicacid agents (siRNA), as shown in Table 7.

TABLE 7 Lipid formulations for siRNA Compound Choles- DPPE- A6 terolDOPE DOPC mPEG(2000) EE* No. (mol %) (mol %) (mol %) (mol %) (mol %) (%)1 25 30 30 10 5 92 2 25 30 25 15 5 91 3 25 30 20 20 5 87 4 25 30 15 25 593 5 25 30 10 30 5 92 6 25 35 15 20 5 93 7 25 35 20 15 5 95 8 30 30 1520 5 87 9 30 30 20 15 5 92 10 35 30 15 15 5 88 *Encapsulation efficiencyfor siRNA.

Example 38

Formulations were prepared for encapsulating a small interfering nucleicacid agents (siRNA).

Lipid nanoparticle formulations were prepared with the followingcompositions:

(Ionizable compound/DOPE/DOPC/Cholesterol/DMPE-PEG) mol %

-   -   50/28/0/21/1    -   50/28/0/20/2    -   26/21/21/31/1.

Characteristics of the nanoparticle formulations are shown in Table 8.The formulations had superior properties for encapsulating the siRNA,and in having small particle size.

TABLE 8 Lipid formulations for siRNA Compound No. Z (ave) nm EE* (%) C2111 95 A6 121 96 A9 97 95 D15 117 96 C24 127 98 DD 132 92 E4 131 94 AA120 90 *Encapsulation efficiency for siRNA.

Example 39

Formulations were prepared for encapsulating a small interfering nucleicacid agents (siRNA).

Lipid nanoparticle formulations were prepared with the followingcompositions:

(Ionizable compound/DOPE/Cholesterol/DMPE-PEG) mol %

-   -   50/28/21/1.

Serum stability and EC50 of nanoparticle formulations were measured andare shown in Table 9. The formulations were capable of encapsulating thesiRNA, and retaining stability in human serum.

TABLE 9 Lipid formulations for siRNA EC50 GFP t(½) (hr) Compound pKasiRNA (nM) human serum 37 C. D23 4.8 23 D24 >200 D25 3.7, 9.5 D26 5.4D27 4.2, 8.9 151 D28 4.5, 7.3 118 D29 6.7 >200 D30 >200 D31 >200 D32 5.461 D33 D34 6.0 91 D35 6.1 21 D36 D37 15 A4 5.8 93 D38 5.3 33 D39 >200D40 >200 D41 D42 >200 D43 10.1  >200 D44 3.7, 9.1 26 D45 >200 D46 D47D48 D49 D50 >200 D51 4.1, 9.1 39 D52 9.7 >200 D53 D54 8.2 >200 D55 4.514 D56 >200 D57 123 D58 D59 D60 D61 D62 9.4 92 D63 D64 8.8 41 D65 >200C3 6.9 >200 D66 >200 D67 6.6 >200 D68 5.7 >200 C2 33 >200 D69 D70 3.0130 D71 42 D72 4.7 >200 D73 >200 D74 4.3 85 F6 9.3 97 AA 7.4 22 81 D759.1 108 D76 4.5 B8 4.3 D77 7.5 D78 5.6 136 D79 D80 5.6 >200 D81 5.9 >200D82 D83 4.8, 7.2 47 D84 6.4 80 A23 E37 9.2 79 A9 7.4 31 56 D85 >200 D867.7 >200 D87 >200 D88 82 F5 9.4 33 59 F8 72 F7 >200 F9 >200 D89 A6 6.311 A5 6.1 6.9 D90 AB 6.9 7.4 A7 7.6 35 A8 D91 D92 D93 D94 E38 D95 D966.0 5.1 DD 5.1 E4 2.7 D97 D98 C25 E39 D99 D9A 60 C24 92 D9B D9C D9D <50CA <50 D1

Example 40

Example formulations are prepared for encapsulating a small interferingnucleic acid agents (siRNA).

Lipid nanoparticle formulations are prepared with the followingcompositions:

(Ionizable compound/DOPE/Cholesterol/DMPE-PEG) mol %

-   -   50/28/21/1.

Serum stability of a nanoparticle formulation is measured and is shownin Table 10. The formulations are capable of encapsulating the siRNA,and retaining stability in human serum.

TABLE 10 Lipid formulations for siRNA t(½) (hr) Compound human serum 37C. C24 >50 AB >50 A6 >50 B8 >50 A23 >50 A5 >50 A7 >50 A8 >50 E38 >50DD >50 E4 >50 C25 >50 E39 >50

The embodiments described herein are not limiting and one skilled in theart can readily appreciate that specific combinations of themodifications described herein can be tested without undueexperimentation toward identifying nucleic acid molecules with improvedRNAi activity.

All publications, patents and literature specifically mentioned hereinare incorporated by reference in their entirety for all purposes.

It is understood that this invention is not limited to the particularmethodology, protocols, materials, and reagents described, as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention. It will be readilyapparent to one skilled in the art that varying substitutions andmodifications can be made to the description disclosed herein withoutdeparting from the scope and spirit of the description, and that thoseembodiments are within the scope of this description and the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprises,” “comprising”,“containing,” “including”, and “having” can be used interchangeably, andshall be read expansively and without limitation.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. For Markush groups, those skilled in theart will recognize that this description includes the individualmembers, as well as subgroups of the members of the Markush group.

A compound, molecule or composition of this invention may have an ionicform for which the corresponding counterion or counterions are notshown. A person of skill in the art will immediately understand that thecounterion or counterions will exist as necessary. Examples ofcounterions include alkali metal ions, Cl⁻, and pharmaceuticallyacceptable counterions.

For example, when a list of examples or components is given, such as alist of compounds, molecules or compositions suitable for thisinvention, it will be apparent to those skilled in the art that mixturesof the listed compounds, molecules or compositions may also be suitable.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever.

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose.

What is claimed is:
 1. A compound comprising the structure shown inFormula I:

wherein R¹ and R² areR¹=CH₂(CH₂)_(n)OC(═O)R⁴R²=CH₂(CH₂)_(m)OC(═O)R⁵ wherein n and m are each independently from 1 to2; R⁴ and R⁵ are independently for each occurrence a C(12-20) alkylgroup, or a C(12-20) alkenyl group; R³ is selected from

wherein each R⁶ is independently selected from alkyl, hydroxyl,hydroxyalkyl, alkoxy, alkoxyalkoxy, and aminoalkyl; each R⁸ isindependently selected from alkyl, and aminoalkyl, and any two R⁸ mayform a ring; each R¹⁰ is independently selected from alkyl, hydroxyl,hydroxyalkyl, alkoxy, alkoxyalkoxy, and aminoalkyl; q is from zero tofour; p is from 1 to 4, wherein the number of double bonds of R⁴ and R⁵is 3 or more when R⁸ is methyl.
 2. The compound of claim 1, wherein eachalkyl of R⁶, R⁸, and R¹⁰ is C(1-6)alkyl, each hydroxyalkyl of R⁶ and R¹⁰is hydroxyl[C(1-6)alkyl], and each aminoalkyl of R⁶, R⁸, and R¹⁰ isamino[C(1-6)alkyl].
 3. The compound of claim 1, R4 and R5 areindependently for each occurrence a C(14-18) alkyl group, or a C(14-18)alkenyl group.
 4. The compound of claim 1, wherein n and m are
 1. 5. Thecompound of claim 1, having a structure selected from


6. A composition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 7. The composition of claim 6, wherein thecomposition comprises nanoparticles.
 8. A pharmaceutical compositioncomprising a compound of claim 1, an active agent, and apharmaceutically acceptable carrier.
 9. The composition of claim 8,wherein the compound is from 15 mol % to 40 mol % of the lipids of thecomposition.
 10. The composition of claim 8, wherein the compositioncomprises nanoparticles.
 11. The composition of claim 8, wherein theactive agent is one or more RNAi molecules.
 12. A composition for use indistributing an active agent for treating a condition or disease in asubject, the composition comprising a compound of claim 1, a structurallipid, a stabilizer lipid, and a lipid for reducing immunogenicity ofthe composition.
 13. The composition of claim 12, wherein the activeagent is one or more RNAi molecules and the composition comprisesnanoparticles that encapsulate the RNAi molecules.
 14. The compound ofclaim 1, wherein R⁴ is a C17 alkenyl group having two double bonds; R⁵is a C17 alkenyl group having two double bonds; n is 1; m is 1; R³ isselected from

each R⁸ is methyl; and q is 1.